Re: [Histonet] Coverslipping mystery

[Adam Boanas]

Hello again,

Thought I might offer an update on the coverslipping issue as it might be of 
use in future.
I ran a test last week of manual coverslipping using blank charged and 
un-charged slides and using DPX and Pertex as the mountant. I also used 2 
methods of application.
1) Mountant applied using plastic Pasteur pipette
2) Mountant applied using aluminium screw cap tube.
Following immersion in xylene for 5 mins the coverslips were applied. From 
viewing this morning, all slides were clear with the exception of those 
coverslipped using DPX applied with the Pasteur. In each case, these slides had 
the 'parched earth' artefact having been left to dry over the weekend.
I suspect that the DPX has had a reaction with the plastic of the pipette 
during application and the artefact is caused by residual `molten` plastic from 
the pipette that only reveals itself over time.
Does this sound plausible? No problem with the pertex and pipettes (which is 
what I've used for years with no issue)
Thanks
Adam

-Original Message-
From: Caroline Miller [mailto:mi...@3scan.com] 
Sent: 11 July 2015 15:18
To: John Kiernan
Cc: Adam Boanas; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Coverslipping mystery

I really like DPX, although funnily enough we used cytoseal in my lab in London 
but always called it the DPX! I think I remember by boss telling me about the 
bad DPX time.

When I moved to the USA the lab I started in had a bottle of DPX and i loved 
it! I always decant some of the DPX into a 100ml glass bottle, put in a plastic 
squeeze pipette and then screw a lid on it to stop it drying out (with the 
pipette still inside) when not in use. Surprisingly the pipette doesn't melt! 
Which is good because I am a recycle freak and i couldn't stand using a new one 
every time I mounted something!

Yours,
mills

Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

 On Jul 10, 2015, at 10:55 PM, John Kiernan jkier...@uwo.ca wrote:
 
 DPX is a polystyrene mounting medium. In principle you can make your own from 
 published recipes. In practice, everyone buys commercial resinous mounting 
 media.
 
 In the 1990s we had trouble similar to what you describe. The commercial DPX 
 was cloudy, and not because of alcohol in our xylene. The Canadian supplier 
 acknowledged the bad DPX and urged us to buy Entellan instead. Entellan is a 
 poly(methacrylate) plastic and is an excellent but expensive mounting medium. 
 Another poly(methacrylate) mountant called CytoSeal was less expensive and 
 also came in a squeeze-easy plastic bottle for delivery onto the slide or 
 coverslip. It's now my routine resious mountant. 
 
 Good DPX returned to the market in the 2000s, but in old-fashioned bottles 
 and not easy to apply to slides or coverslips. 
 
 John Kiernan
 = = =
 On 09/07/15, Adam Boanas  a.boa...@epistem.co.uk wrote:
 Hello,
 
 We are having a problem that is developing into a big issue in our lab and I 
 was wondering if anybody could shed any light on it. Our CV5000 coverslipper 
 has recently started introducing microscopic air bubbles onto the slides 
 during coverslipping. We have been told by our engineer that it is a 
 consequence of the age and use of the motor and that sourcing another for an 
 instrument that old (15yrs) will be v difficult. As such, we have been 
 forced to manually coverslip using DPX and a pipette - manually applying the 
 coverslips to the slide, thus mirroring the action of the coverslipper. This 
 is fine at first and for the next few days the slides look great and very 
 clean. However, after about day 4 -5 days post coverslipping, the slides 
 develop an odd appearance down the microscope which looks like very fine 
 `parched earth / crazy paving` all over the slide - including the section. 
 The excess mountant around the edge of the coverslip also has a very faint, 
 cloudy appearance wh!
  en this occurs. This of course renders the slide un-useable. Does anyone 
 have a clue what this might be down to / how we can stop it?
 We are struggling for ideas with this one! - this occurs with fresh DPX also.
 
 Many thanks
 Adam
 
 Adam Boanas
 Senior Research Associate
 Epistem Ltd
 48 Grafton Street
 Manchester, M13 9XX
 
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[Histonet] Coverslipping mystery

[Adam Boanas]

Hello,

We are having a problem that is developing into a big issue in our lab and I 
was wondering if anybody could shed any light on it. Our CV5000 coverslipper 
has recently started introducing microscopic air bubbles onto the slides during 
coverslipping. We have been told by our engineer that it is a consequence of 
the age and use of the motor and that sourcing another for an instrument that 
old (15yrs) will be v difficult. As such, we have been forced to manually 
coverslip using DPX and a pipette - manually applying the coverslips to the 
slide, thus mirroring the action of the coverslipper. This is fine at first and 
for the next few days the slides look great and very clean. However, after 
about day 4 -5 days post coverslipping, the slides develop an odd appearance 
down the microscope which looks like very fine `parched earth / crazy paving` 
all over the slide - including the section. The excess mountant around the edge 
of the coverslip also has a very faint, cloudy appearance when this o
 ccurs. This of course renders the slide un-useable. Does anyone have a clue 
what this might be down to / how we can stop it?
We are struggling for ideas with this one!  - this occurs with fresh DPX also.

Many thanks
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

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RE: [Histonet] Frozen section artefact

[Adam Boanas]

Hi again,

In your opinion is it better to fix frozen sections prior to storage in -80 or 
following removal from -80 prior to a run?
Thanks
Adam

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Hobbs, Carl
Sent: 25 September 2012 18:37
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Frozen section artefact

Assuming that you are fixing fresh-frozen tissue sections:

Tissue is autolysing.

Use Formalin for 10 mins or 1:1 acetone:alcohol for 10 mins.



Imho, acetone is not a fixative..it's a delipidizer.

5 mins in that mixture is too short a time for the alcohol to be an effective 
coagulant fixer.

However, a longer time in alcohol may well mask your Ag sites.



I see this artefact in acetone fixed frozen sections, often.



When immunostaining for MHCs in muscle, no fixation is required but, if 
DAPI/Hoechst is includednuclear streaming will be seen.



Always try several fixing fluids to get the best results on any new Ab.



 I may be wronghappy to be corrected.



Curious always,



Carl






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[Histonet] Frozen section artefact

[Adam Boanas]

Hello,

I have a puzzling artefact that I can't seem to correct in 7 micron frozen 
sections of rat and mouse Small Intestine. When completing an IHC run, parts of 
the section look great and the staining has worked fine - on other parts of the 
same section, the morphology is ruined by the appearance of several spindly 
striations or smears that run between individual villi and in some cases 
actually cover the majority of the section. Where this occurs the top half of 
the villi do not take up either the antibody staining or the Haematoxylin 
counterstain (which is taken up fine elsewhere). Sometimes the smearing looks 
so bad that it turns the section into a smeary, gloopy mess. From what I can 
see the origin of the smears appears to be the nuclei as I have seen several 
small spindles from the nuclei leading into a larger thread.
We have thought about Lysis, Gut Mucus (have stained PAS to highlight), nuclear 
degredation.
Could it be fixation? We air dry following sectioning and fix in Acetone / 
alcohol 3:1 for 5 mins.
Any ideas would be great! Thanks in advance.
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX



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[Histonet] What is this item called?

[Adam Haberman]

Hello everyone,

I am trying to identify a piece of glassware so that I can order a few more
of it.  It is a thick piece of glass with a large, wide depression in the
middle.  The item I am looking for is thicker and wider than a depression
slide, and I use it as a dissecting dish.  I have included a picture of the
piece I am trying to identify, along with a depression slide for
comparison.  This is probably an item that is not widely used anymore, as
it seems to predate all of the faculty here.  However, it is perfect for
the dissections I do with two pair of forceps under a dissecting
microscope.  It gives me much better working angles than the spot plates I
resort to when I cannot find this item.

If anyone can identify the item or, even better, tell me where I can order
more, please let me know.  Thank you very much.

--Adam

Adam Haberman
Visiting Assistant Professor of Biology
Oberlin College
adam.haber...@oberlin.edu
(440)775-6502
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Re: [Histonet] What is this item called?

[Adam Haberman]

Barry, It is 3 x 1 3/4, and the depression has a 1 1/2 diameter.

Thanks for all the ideas so far.  It isn't a hanging drop slide or a watch
glass.  You can see a picture of it next to a depression slide here:
http://imgur.com/yBwvw
I don't see a better way to send a picture on this list.

--Adam

Adam Haberman
Visiting Assistant Professor of Biology
Oberlin College
adam.haber...@oberlin.edu
(440)775-6502



On Tue, Jul 3, 2012 at 2:27 PM, Long, Florence lon...@labcorp.com wrote:

 Hi Adam,
 Would that be a modification of a hanging-drop slide?
 F. Long

 
 From: histonet-boun...@lists.utsouthwestern.edu [
 histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Adam Haberman [
 adam.haber...@oberlin.edu]
 Sent: Tuesday, July 03, 2012 8:45 AM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] What is this item called?

 Hello everyone,

 I am trying to identify a piece of glassware so that I can order a few more
 of it.  It is a thick piece of glass with a large, wide depression in the
 middle.  The item I am looking for is thicker and wider than a depression
 slide, and I use it as a dissecting dish.  I have included a picture of the
 piece I am trying to identify, along with a depression slide for
 comparison.  This is probably an item that is not widely used anymore, as
 it seems to predate all of the faculty here.  However, it is perfect for
 the dissections I do with two pair of forceps under a dissecting
 microscope.  It gives me much better working angles than the spot plates I
 resort to when I cannot find this item.

 If anyone can identify the item or, even better, tell me where I can order
 more, please let me know.  Thank you very much.

 --Adam

 Adam Haberman
 Visiting Assistant Professor of Biology
 Oberlin College
 adam.haber...@oberlin.edu
 (440)775-6502
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Re: [Histonet] What is this item called?

[Adam Haberman]

Jay, there are no markings on it at all.  They are making it hard for me to
give them money.

--Adam

Adam Haberman
Visiting Assistant Professor of Biology
Oberlin College
adam.haber...@oberlin.edu
(440)775-6502



On Tue, Jul 3, 2012 at 3:32 PM, Jay Lundgren jaylundg...@gmail.com wrote:


 Brilliant way to send a picture.  I 3 interwebs.  Whatever it is, I want
 one.  So it's made of optical quality glass, like a slide or a coverslip?
 Does it have a company's name on it?  I'll bet you it's expensive.

Sincerely,

  Jay A. Lundgren,
 M.S., HTL (ASCP)
































































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[Histonet] Translucent patch

[Adam Boanas]

Hello,

We have recently noticed strange translucent patches that can be seen within 
our 3 micron tissue sections when they are floated out. From the surface of the 
water bath they look like holes within the tissue but when the slides are 
viewed, the tissue is still present. This region of tissue however, creases and 
folds when mounted. The rest of the section looks and behaves perfectly - it is 
only this translucent region that is causing the problem. I have seen this 
region as a small blob but also as a thin streak that runs through the entire 
section. This region can only be seen for the first 7-10 sections taken. After 
this the translucent region gets smaller and disappears. This is making getting 
perfectly flat, artefact free sections of tissue (mouse / rat gut / liver) 
difficult. Does anyone have any idea what this could be? My thoughts are a 
possibly processing issue - currently process without vacuum wax infiltration 
or an embedding issue. Our metal moulds were cleaned with a methanol based 
para-release spray about 2 months ago by mistake - we have had subsequent 
tissue spreading issues as a result. Could traces of this affect the cutting 
surface of the tissue causing this patch?
Any ideas would be great,
Many thanks,
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

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[Histonet] Whiskers

[Adam Boanas]

Hello,

I am currently sectioning individual rat whiskers and follicles and am having 
some trouble obtaining clean sections from within the centre of the whisker 
(and intact bulb) without the brittle hair falling out of the block or 
splintering. Does anyone know of a method that I could use to soften the 
keratin prior to embedding? I am thinking 10% Sodium Hydroxide or 5% Ammonium 
Hydroxide but do not really know about appropriate timings for such a small 
tissue.
Many thanks,
Adam

Adam Boanas
Senior Research Associate
Epistem Ltd
48 Grafton Street
Manchester, M13 9XX

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[Histonet] RE: KP Markers!

[Adam Harris]

If you would like to obtain KP Markers, they are now being carried by Sensor
Health, inc. in Cambridge, Ontario, Canada. If you have any questions you
can contact Adam Harris at 888-777-7080 or 519-241-2194. You can check out
their website at www.sensorhealth.com 



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[Histonet] Specimen Clamps Leica

[Adam Hacking]

Hi I'm looking for some used specimen clamps for a Leica RM 2255. I'm 
interested in any clamp that is for holding round or irregularly shaped 
specimens.

Thanks

Adam 
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[Histonet] So long, and thanks for all the help

[Adam .]

Dear Histonet:

For the past few years, I have been working on my PhD. I realized very early
on in my research that immunostaining of bone would be absolutely necessary
for my project, although the initial attempts in our lab to do that were not
particularly satisfying. I tried talking to various local people, who gave
me some good advice, but it was not until I found HistoNet that I really
began to understand how to attack such a frustrating problem. I began my
project with my PI telling me that staining of bone would be impossible. I
wish I could take credit for proving him wrong, but in reality, I'm showing
him the results I get when I follow all the advice that I read here.

I have found all members of HistoNet so incredibly knowledgeable, patient,
friendly, and funny. It really is amazing that such a great online community
could be gathered, focusing on such a technical field (I've subscribed to
some other forums where people aren't quite as nice). I've thoroughly
enjoyed reading all the discussions ranging from advice on how to decalcify
coral to silly jokes and stories passed around.

In particular I would like to thank two members of this group: Andrea Hooper
and Gayle Callis. They have corresponded with me extensively both on and off
the board and have been incredibly helpful in accomplishing my work. I
honestly do not think my thesis project could have been finished without
their assistance. If we ever physically cross paths, I will totally buy you
two a drink and a meal.

Fortunately (unfortunately?), I have to move on with my life. I am an MD/PhD
student, so in a few days I need to return to the hospital working long
hours, frantically trying to relearn what I forgot years ago. Alas, my
friends tell me I will barely have time to sleep, and certainly not enough
time to read HistoNet. I am thus unsubscribing from the list until I need to
do such staining again, when I am back in research. Can you please
unsubscribe me?

I'm joking. I've read enough of those e-mails to know how to unsubscribe
properly.

Anyhow, I would like to reiterate my thanks to the group. You are an amazing
group of people.

Sincerely,
Adam
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Re: [Histonet] protocol for in situ RT_PCR

[Adam .]

I've used 10% zinc buffered formalin from Anatech (no alcohol) on mouse
bones with good results. It's possible that the alcohol is denaturing your
antigens of interest. Is there a reason you need to use alcoholic formalin?

Adam

On Tue, Aug 16, 2011 at 7:45 AM, Liang, Frank fli...@wellstatoc.com wrote:

 Hello,

 Does anyone have used alcoholic zinc-formalin to fix mouse tissues? We
 tried  a few times with this fixative for immunohistochemical staining, it
 did not work, but when switched back to 4% paraformaldehyde, the
 immunostaining worked. We like alcoholic zinc-formalin as it gives better
 morphology. Any suggestions?

 Thanks,

 Frank

 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu [mailto:
 histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Louise Renton
 Sent: Tuesday, August 16, 2011 4:17 AM
 To: Barone, Carol; Histonet
 Subject: Re: [Histonet] protocol for in situ RT_PCR

 hi could you add me on too pelase?

 On Mon, Aug 15, 2011 at 11:09 PM, Barone, Carol cbar...@nemours.org
 wrote:

  Anyone got a hot protocol for In situ RT-PCR? Haven't done one in 5
  years (on my Perk and Elmer)...Know there must be some great new
  reagents and kits out there...opinions? Send to cbar...@nemours.org. Thx
  CB
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 --
 Louise Renton
 Bone Research Unit
 University of the Witwatersrand
 Johannesburg
 South Africa
 +27 11 717 2298 (tel  fax)
 073 5574456 (emergencies only)
 There are nights when the wolves are silent and only the moon howls.
 George Carlin
 No trees were killed in the sending of this message.
 However, many electrons were terribly inconvenienced.
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Re: [Histonet] Decalcification of bone

[Adam .]

Hi Rachael,

I work with mouse bones on a regular basis, and I assure you that they are
incredibly difficult to work with, but with practice you can get decent
sections. It would be useful if you gave us a bit more information. What
kind of bones are you decalcifying? How are they fixed? How are you
currently decalcifying? Are you paraffin embedding or cutting frozen
sections? What is exactly happening when you're cutting the bones that gives
you poor quality? What is your end goal (IHC, IF, HE)?

Adam

On Thu, Aug 11, 2011 at 3:10 AM, Rachael Glebocki 
rachael.glebo...@nottingham.ac.uk wrote:

 Dear Histonet users,

 I was wondering if anyone has a operation procedure for bone
 decalcification that works. I am having no joy in decalcifying the bone and
 making a good slide from it.

 Thank you for your time.

 Rachael Glebocki
 Teaching Technician
 School of Veterinary Medicine  Science
 University of Nottingham

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[Histonet] Cutting frozen decalcified mouse bones

[Adam .]

Hi all,

I had a very difficult time cutting my very precious triple transgenic
frozen decalcified mouse bones this morning. As I cut into them, the
sections scrunched up into a mess of OCT pretty much as soon as the blades
hit the block. These were 4% PFA perfused and post-fixed for 24 hours,
decalcified with 14% EDTA using weight loss/weight gain as an endpoint,
cryoprotected overnight in 30% sucrose, embedded in OCT using liquid
nitrogen cooled isopentane vertically (i.e. I was cutting from one growth
plate to the other), and cut at 7 uM. Changing to a new blade didn't help.
Changing the cutting temperature from -10C to -15C didn't help. Changing the
cutting speed didn't help. I can't change the cutting angle without a
special tool, and this isn't my device so I don't want to mess with it.

The only way I could get reasonable sections was to use a tape transfer
system, which seemed like overkill to me, but I need these sections ASAP.
Ideas? I have a whole bunch more bones I need to cut over the next few
weeks...

Thanks,
Adam
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Re: [Histonet] Light microscopy to get orientation for fluorescent

[Adam .]

Try differential interference microscopy (DIC). Many fluorescent scopes will
have this as an option. It will give you faint outlines of the cells.

Adam

On Sun, Jul 17, 2011 at 1:01 AM, Otto Strauss olstra...@gmail.com wrote:

 I am trying to do fluorescent immunohistochemistry on liver tissue.
 Unfortunately because the liver is such a homogenous tissue it is difficult
 to get an appropriate orientation with just fluoroscopic views.

 I was wondering if there is a way of staying with non fluorescent dyes(like
 HE) so that I can get a picture with light microscopy, to have orientation
 within the liver when viewing it with fluorescent microscopy?

 Regards.

 Otto
 Auckland
 NZ

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Re: [Histonet] pan B-cell marker for frozen mouse tissue

[Adam .]

B220 is a pretty good B-cell marker, although you're right that it's not
restricted to the B-cell lineage. It's also expressed in some T-cells and NK
cells, for example. Cells that are double positive for B220 and IgG or IgM
are B-cells almost exclusively. CD19 is a good marker for mature B-cells,
although pre-pro B cells do not express CD19. If you're looking at any organ
other than bone marrow or spleen, there shouldn't be many pre-pro B cells
and you can probably just use CD19.

Adam

On Mon, Jul 11, 2011 at 1:37 PM, Michele Wich mw...@7thwavelabs.com wrote:

 I am trying to find the most appropriate pan B-cell marker for IHC on
 frozen mouse tissue. I know that CD45R/B220 used to be the most commonly
 used, but recent publications suggest PAX5 or CD19 as more restricted to
 the B-cell lineage. Does anyone have any other suggestions, or are these
 generally what people are using these days?

 Thanks for any info.





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[Histonet] Sad news

[Adam .]

Dear histology friends,

I know this is a fairly close community, so I thought I should inform you
that our local histotech, Patricia Pat Keller, passed away a few weeks
ago, suddenly and unexpectedly. I apologize for the delay in relaying this
information, but I wanted to wait until I confirmed the news with a
coworker. From what I've heard secondhand, she came home from work one day,
told her son she wasn't feeling well, went to sleep, and never woke up. I
didn't know Pat that well, but I did spend one afternoon with her as she
taught me to cut sections of mouse bone. In terms of the technical expertise
she provided to the scientific community, she will definitely be missed. For
those of you who knew her personally, I'm sure she will be missed much more.

Adam
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[Histonet] Flk2 / Flt3 / CD135 on mouse bone

[Adam .]

Hi all,

One of my fellow graduate students is trying to perform immunofluorescence
on PFA fixed, EDTA decalcified mouse bone for Flk2 (aka Flt3 or CD135). So
far, he hasn't had any luck on either paraffin embedded or frozen sections
using HIER. Has anyone done this successfully? I know the ideal way to
perform IF on bone is using a tape-transfer system, but, alas, we don't
quite have that working yet.

Thanks,
Adam
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Re: [Histonet] Flk2 / Flt3 / CD135 on mouse bone

[Adam .]

Hi Ray,

That is a great idea. We are indeed looking for Flk2 expression on
hematopoietic progenitors cells, but alas, we are looking at these cells in
relationship to bone elements such as osteoblasts, so we need to do it on
intact bone.

Adam

On Mon, Jun 27, 2011 at 8:43 PM, koelli...@comcast.net wrote:

 Adam/his fellow grad student,



 I could be somewhat off not being up to date with current literature.
 Sorry.  But used to look for Flk2/Flt3/CD135 in murine bone marrow and not
 the bone itself.  In immature hematopoeitic progenitor cells.  Along with
 looking in thymus, etc.  But for something like femurs, I *removed* the
 bone marrow, and there are some slick ways to do it without disrupting the
 core architecture too much.  Could take it out and obviously flow
 (cytometry) the cells.  But could also get out a fairly intact core and
 freeze it, or paraffin process/section it or glycol methacrylate section it
 and worry very little about the minute amount of trabecular bone in murine
 bone marrow. Depending on age, etc of course.



 So unless for the specific needs of the project call for looking actually
 for staining within the cortical bone, I'd take that problem out of the
 picture by getting the bone marrow out and then not having to worry  about
 decalcification and tape transfer and things like that.  Makes IHC staining
 a lot easier in my experience but  perhaps there are those out there who
 have worked out whole, intact mouse bone CD135 staining.



 Ray



 Ray Koelling

 PhenoPath Labs

 Seattle WA

  --

 *From: *Adam . anonwu...@gmail.com
 *To: *histonet@lists.utsouthwestern.edu
 *Sent: *Monday, June 27, 2011 5:49:23 PM
 *Subject: *[Histonet] Flk2 / Flt3 / CD135 on mouse bone


 Hi all,

 One of my fellow graduate students is trying to perform immunofluorescence
 on PFA fixed, EDTA decalcified mouse bone for Flk2 (aka Flt3 or CD135). So
 far, he hasn't had any luck on either paraffin embedded or frozen sections
 using HIER. Has anyone done this successfully? I know the ideal way to
 perform IF on bone is using a tape-transfer system, but, alas, we don't
 quite have that working yet.

 Thanks,
 Adam
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Re: [Histonet] antigen retreval method-mouse femour

[Adam .]

Hi Mani,

This is a very common problem for bone IHC, and there is no perfect
solution. Here is what I do: after cutting the paraffin embedded, fixed and
decalcified sections, I dry the slides sitting flat in a 37C oven for 5 or
more days. The longer you dry them, the better your results. Then I retrieve
with citrate buffer pH 6.0 for 10 mins at 95C. The bone generally stays
intact, although the morphology is rarely as nice as unretrieved sections.
Some people swear by other antigen retrieval methods that don't involve
heat, but I've never managed to get them to work.

Adam

On Sun, May 29, 2011 at 3:52 PM, mani kandan coralm...@yahoo.co.in wrote:


 hai,
 i am working with mouse femours, can any one have a experience in
 immunohistochemistry on mouse femours,because after antigen retreval they
 cancellous bone not looks good,morphology of cells also changed, can anyone
 tell me about good antigen retrieval method.

 M.Manikandan,
 Researcher,
 Stemcell unit,
 King Saud university,
 Riyadh,KSA
 +966552012697
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[Histonet] Xylene re-use SOP

[Adam .]

Hi all,

Our small research lab recently has been investigated by a very aggressive
environmental health and safety inspector, and she asked us to write up the
standard of practice for any chemicals that we re-use, including all the
chemicals we re-use for deparaffinization and dehydration and rehydration of
slides. We currently keep a few bottles of xylene and graded ethanol that we
dispense into staining racks used for deparaffinizing and rehydration of
sections. We pour the bottles back into their containers once we're done
with them and reuse them until it has a lot of paraffin detritus in it. We
do this all by hand.

EHS wants us to do the following:

If a container is labeled in process or recycled be sure to have an SOP
written up describing what is being done and what is meant by recycled (in
this case a solution is being used again).  Be sure to say at what point the
solution is waste and then how it is managed after that (properly labeled
and disposed). Don't label containers “waste” or “used” unless it is labeled
with a properly filled out yellow hazardous waste sticker provided by EHS.

I have no idea what they're looking for, but they've gotten quite strict in
enforcing all sorts of vague regulations. Any suggestions are welcome.

Thanks,
Adam
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Re: [Histonet] GFP labelled cells after decalcification...are they still labelled?

[Adam .]

Hi James,

I do this all the time in mouse bones. I've found that EGFP is very robust,
and it can survive fixation with PFA and Zn buffered formalin, as well as
decalcification with EDTA or formic acid, followed by paraffin embedding.
However, the endogenous fluorescence is highly quenched by this process and
barely detectable over background if at all. In order to detect it post
embedding, I use the chicken anti-GFP polyclonal from Abcam, and a
fluorophore conjugated donkey anti-chicken IgY with very good results. If
you want endogenous fluorescence, you can fix the bones in 4% PFA and
decalcify using EDTA, and use frozen sections.

I hope this helps,
Adam

On Thu, Mar 31, 2011 at 12:42 PM, James Hart jameshar...@gmail.com wrote:

 Hi All

 A first time user of Histonet here -with a quick question for research
 histologists...

 Has anyone ever attempted to locate GFP expressing cells in
 bone-marrow sections after decalcification and paraffin embedding? (I guess
 my question really is: Does the decalcification process destroy or weaken
 the GFP signal)

 Any advice would be greatly appreciated!

 Regards

 James

 Dr James Hart BVSc MS
 Comparative Orthopaedics, Cornell University
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[Histonet] Mouse CD45 staining of bone marrow

[Adam .]

Hi all,

I have been attempting to do mouse CD45 immunofluorescence on frozen, fixed
decalcified mouse sections and have an odd problem. I am using the
monoclonal rat anti-mouse CD45 (clone 30F-11 from BD) and get very nice
staining along the cell surface of many cells. However, only about 20% of
the nucleated cells stain with CD45.

By FACS,  90% of the cells stain with CD45 using the same clone. To be
fair, that is with RBC lysis, but the RBCs should not be nucleated anyhow.
The antibody comes at 62.5 ug / mL, and I am using the antibody at a 1:1000
dilution in TBS-T. I found that at the recommended1:20 titer, nothing
stained above background. I only began to get staining at  1:100,
suggesting to me there may have been some steric hindrance going on.
Although this clone is supposed to work on FFPE sections, I have been unable
to get any staining at all using even with aggressive HIER.

I was wondering if anyone has IHC or IF photos of CD45 staining in mouse
bone marrow so I know what it should look like. Any suggestions are welcome.

Thanks,
Adam

The specifics:
1) Fix the bones overnight in 4% PFA at 4C
2) Decalcify in EDTA for 3 days at 4C
3) Cryoprotect in 30% sucrose overnight at 4C
4) Embed in OCT using liquid nitrogen cooled 2-methylbutane
5) Section using Cryojane tape transfer system. I have also tried this with
a regular cryotome with similar results.
6) Block in 10% donkey serum, then avidin and biotin
7) Incubate with primary antibody overnight at 4C. Wash.
8) Incubate with biotinylated donkey anti-rat F(ab)2 (1 ug / mL) at room
temperature for 1 hr. Wash.
9) Incubate with strepavidin Dylight 594 (1 ug / mL) at room temperature for
1 hr. Wash.
10) Mount with Prolong Gold anti-fade with DAPI
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[Histonet] tungsten carbide knives

[Adam Hacking]

Hi 
 
If anyone has any used tungsten carbide knives that they no longer need please 
let me know.  These should be in good shape and suitable for sharpening. I'm 
happy to pay a resonable price for them.
 
Thanks
 
Adam

--- On Thu, 2/10/11, histonet-requ...@lists.utsouthwestern.edu 
histonet-requ...@lists.utsouthwestern.edu wrote:


From: histonet-requ...@lists.utsouthwestern.edu 
histonet-requ...@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 87, Issue 20
To: histonet@lists.utsouthwestern.edu
Date: Thursday, February 10, 2011, 1:51 PM


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When replying, please edit your Subject line so it is more specific
than Re: Contents of Histonet digest...

Today's Topics:

   1. RE: QIHC (Houston, Ronald)
   2. RE: CPT code 88363 (Mike Pence)
   3. RE: QIHC (Stephanie Rivera)
   4. Cassette labeling (Nancy Schmitt)
   5. HT Position in Oklahoma City (Gaiser, Marcia)
   6. Supervisory/Lead Job in Fort Myers, FL (Alyssa Peterson)
   7. RE: CPT code 88363 (Richard Cartun)
   8. Re: QIHC (Mark Tarango)

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Re: [Histonet] Re: Banana smelling chemical in our formalin

[Adam .]

That wasn't formalin. It was Histologie for Men, the new banana-scented
cologne for histotechs.

But seriously, I guess it's formalin ethyl acetate, which smells vaguely
banana-like and is used for extraction of parasites from stool samples.

Adam

On Mon, Jan 24, 2011 at 4:56 PM, Patrick Laurie foreig...@gmail.com wrote:

  Histonet,
 
  We get a number of our specimens from outreach clients.  We received back
  from one of our sites a jug of Formalin to be recycled.  However when
 it
  was opened up it had a strong odor of bananas.  I know we shouldn't smell
  the chemicals, but luckily this was caught before it was put through our
  recycler.  I was wondering if anyone might have an idea what this could
 be?
  Is there a manufacturer that adds an odor (like bananas) to their
 formalin?
  Is it a strange solvent?  The idea of it being amyl acetate has been
 bounced
  around, but I cannot see a histologic use for such a chemical.
 
  Thanks for your input.
 
  --
  Patrick Laurie HT(ASCP)QIHC
  CellNetix Pathology  Laboratories
  1124 Columbia Street, Suite 200
  Seattle, WA 98104
  plau...@cellnetix.com
 
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Re: [Histonet] necropsy on freezed animal samples

[Adam .]

I recently had an animal from a previously undescribed mouse model die
suddenly during the weekend. Since I couldn't send it off to necropsy, I
stashed it in the refrigerator until Monday morning. According to the person
who read the histology Post-mortem degeneration limited evaluation of most
tissues.

So I would agree from experience that this is not a good idea.

Adam

On Fri, Jan 21, 2011 at 10:21 AM, Geoff McAuliffe mcaul...@umdnj.eduwrote:

 If the tissue is not fixed soon after death the microscopic morphology will
 be terrible, no matter whether the animal is refrigerated or frozen. The
 person suggesting refrigeration/freezing is (obviously) not familiar with
 histological technique.
 As someone else on the list suggested, better planning is needed for the
 harvesting of tissues for microscopy.

 Geoff


 On 1/21/2011 5:05 AM, krishna_adhik...@mail.com wrote:


 Dear Group Members,
 I have a strange query, One person suggested, if the animals for necropsy
 are more and one can not handle the load of necropsy  on the same day.
 Then simply after sacrificing the animals refrigerate the dead animals to
 reduce the autolysis process and then proceed with the actual necropsy
 procedures.
  is it possible to to the necropsy procedures on the freezed animals
 samples for further histopathology evaluation?
 expert comments are suggested.
 Regards
 krishna

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 --
 --
 **
 Geoff McAuliffe, Ph.D.
 Neuroscience and Cell Biology
 Robert Wood Johnson Medical School
 675 Hoes Lane, Piscataway, NJ 08854
 voice: (732)-235-4583
 mcaul...@umdnj.edu
 **




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[Histonet] Contract Histo Tech Needs in South Carolina

[Adam Schultz]

Hello Everyone!!

I am looking for Two Histotechnologists to perform various routine duties 
and/or highly complex analysis (immunoperoxidase and cutting tissue) within the 
Histology Section of Pathology and Laboratory Medicine at a hospital in 
Columbia, SC.  Work hours are 8a-4:30pm M-F, no weekends, no holidays.  
Contract will go for 17 weeks in length with possibility of extension.  Please 
contact Adam Schultz @ (813) 371-3427 or Kevin Lucania @ (813) 371-5174.  They 
can both also be reached toll free at 888-800-1855.

***Note - Minimum two years experience required to apply***

Thank you,

Adam Schultz
TravelMax Medical Professionals
813-371-3427
adsch...@travmax.commailto:adsch...@travmax.com



Confidentiality Statement: The information contained in this facsimile/email 
transmission is privileged and confidential and is intended only for the use of 
the recipient listed above. If you are neither the intended recipient or an 
employee or agent of the intended recipient responsible for the delivery of 
this information, you are hereby notified that the disclosure, copying, use or 
distribution of this information is strictly prohibited. If you have received 
this transmission in error, please notify us immediately to arrange for the 
return of the transmitted documents or to verify their destruction.
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[Histonet] Toluidine blue staining of CFU-F

[Adam .]

Hi all,

I'm looking for a protocol to counterstain mouse CFU-F grown in tissue
culture**. The protocols I've found just say they counterstained with this
dye, don't say what it's dissolved in, sometimes list the percentage of it,
and never list the time they stained for. The only full protocol I found was
for staining of mast cells in sections, but that's dissolved in 70% ethanol
and very acidified. I'm not sure that's what I want.

Has anyone done this before?

Thanks,
Adam
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[Histonet] Job opening for a Histology Manager at HealthTronics in Augusta, GA 30901

[Smith, Adam]

Hello, my name is Adam, I am a recruiter at Adecco Medical  Science and I have 
an opening for a Histology Manager in Augusta, GA 30901.  This position will 
fill quickly so if you are interested please reach out to me as soon as you 
can.  I have listed the job description and required qualifications about the 
position below.  If you are not interested please feel free to pass this email 
along to anyone who may like to apply for this position. I look forward to 
speaking with you

Please send me 2 - 3 professional references and an updated resume

Job Details:

Position title:  Histology Manager
Start date: ASAP
Duration: Direct Hire
Location: Augusta, GA 30901
Hours: 1st shift

Job Details

Reports to the Director of Laboratory Operations and is responsible for 
coordinating and directing activities of assigned section and subordinates 
engaged in performing routine and non-routine histology procedures. In doing 
so, ensures quality and quantity standards relating to the delivery of 
laboratory services are achieved by assuring results.


-Knowledge of Science and Mathematics normally acquired through completion of 
an Associate's Degree.
-Active/current certification as a Histotechnologist (HT or HTL) by the Board 
of Registry of the American Society of Clinical Pathologists is required.
-Five years experience as an HTIII to include 2 years of management experience 
or 3-5 years experience as an HTIII and completion of internal manager training 
program, or equivalent work experience in a pathology lab setting.

SUMMARY OF PURPOSE:

Reports to the Laboratory Manager and is responsible for coordinating and 
directing activities of assigned section and subordinates engaged in performing 
routine and non-routine histology procedures. In doing so, ensures quality and 
quantity standards relating to the delivery of laboratory services are achieved 
by assuring results.

ESSENTIAL FUNCTIONS:
1.  In collaboration with the Laboratory Manager plans, coordinates, 
monitors and oversees work of assigned section and subordinate personnel 
engaged in performing histology procedures needed to obtain data for disease 
diagnosis and treatment.
2.  Accomplishes, or effectively recommends the following:
*Prepares schedules to assure adequate coverage.
*Assigns work
*Counsels employees on work or working relationships
*Arranges and/or conducts job training, continuing education.
*Approves time worked
*Completes employee orientation for assigned section.
*Interviews, hires, and evaluates the performance of and, when 
necessary, disciplines and discharges subordinate supervisory personnel.
*Approves hiring and, when necessary, discharge recommendations of 
subordinate personnel and assists in resolving complex employee relations 
matters.
3.  Ensures all technical procedures are performed correctly. Procedures 
with higher priority are completed first, equipment has proper maintenance 
performed and kept in good working order.
4.  Reviews and evaluates new products, equipment.
5.  Evaluates and implements new procedures as necessary.
6.  Makes supply decisions with sufficient accuracy so no supplies run out 
or remains beyond the expiration date.
7.  Responsible for daily quality control and quality assurance to include 
establishing training and maintaining Q.C. and Q.A  protocol with subordinate 
staff.
8.  Coordinates monthly QA and QC data.
9.  Ensures that laboratory section meets all safety and other regulatory 
requirements.
10.  Effectively manages technical aspects of hospital accounts
11.  Gathers information and carries through to completion assigned projects 
while adhering to established time frames.
12.  Prepares periodic reports as necessary.
13.  Assures activities of assigned section are coordinated to insure quality 
patient care and economics of operation.
14.  Participates in continuing education as required.
15.  Consults with LIS Administrator to troubleshoot LIS problems and 
enhancements.
16.  Participates in the development of assigned section and department wide 
policies and procedures.
17.  Maintains all section records relating to operation such as quality 
control and productivity statistics, incident reports and the like.
18.  Reviews and summarizes records and prepares reports for management review.
19.  Develops and maintains cooperative working relationships with physicians 
and various other Healthtronics employees.
20.  Provides advice and direction to subordinates for technical problems 
encountered. Explains and demonstrates appropriate techniques or methods as 
necessary.
21.  Performs routine procedures in achieving workload demands.
22.  Maintains knowledge of current trends and developments in the field of 
expertise.
23.  Performs other duties as assigned

POSITION REQUIREMENTS:

Education and formal training:
Knowledge of Science and Mathematics normally acquired through

[Histonet] I would like to post this mesage on to all subscribers on histonet

[Smith, Adam]

Job opening for a Histology Manager in Augusta, GA 30901

Hello, my name is Adam, I am a recruiter at Adecco Medical  Science and I have 
an opening for a Histology Manager in Augusta, GA 30901.  This position will 
fill quickly so if you are interested please reach out to me as soon as you 
can.  I have listed the job description and required qualifications about the 
position below.  If you are not interested please feel free to pass this email 
along to anyone who may like to apply for this position. I look forward to 
speaking with you

Please send me 2 - 3 professional references and an updated resume

Job Details:

Position title:  Histology Manager
Start date: ASAP
Duration: Direct Hire
Location: Augusta, GA 30901
Hours: 1st shift
Pay:  28.00 max


Job Details

Reports to the Director of Laboratory Operations and is responsible for 
coordinating and directing activities of assigned section and subordinates 
engaged in performing routine and non-routine histology procedures. In doing 
so, ensures quality and quantity standards relating to the delivery of 
laboratory services are achieved by assuring results.


-Knowledge of Science and Mathematics normally acquired through completion of 
an Associate's Degree.
-Active/current certification as a Histotechnologist (HT or HTL) by the Board 
of Registry of the American Society of Clinical Pathologists is required.
-Five years experience as an HTIII to include 2 years of management experience 
or 3-5 years experience as an HTIII and completion of internal manager training 
program, or equivalent work experience in a pathology lab setting.

SUMMARY OF PURPOSE:

Reports to the Laboratory Manager and is responsible for coordinating and 
directing activities of assigned section and subordinates engaged in performing 
routine and non-routine histology procedures. In doing so, ensures quality and 
quantity standards relating to the delivery of laboratory services are achieved 
by assuring results.

ESSENTIAL FUNCTIONS:
1.  In collaboration with the Laboratory Manager plans, coordinates, 
monitors and oversees work of assigned section and subordinate personnel 
engaged in performing histology procedures needed to obtain data for disease 
diagnosis and treatment.
2.  Accomplishes, or effectively recommends the following:
*Prepares schedules to assure adequate coverage.
*Assigns work
*Counsels employees on work or working relationships
*Arranges and/or conducts job training, continuing education.
*Approves time worked
*Completes employee orientation for assigned section.
*Interviews, hires, and evaluates the performance of and, when 
necessary, disciplines and discharges subordinate supervisory personnel.
*Approves hiring and, when necessary, discharge recommendations of 
subordinate personnel and assists in resolving complex employee relations 
matters.
3.  Ensures all technical procedures are performed correctly. Procedures 
with higher priority are completed first, equipment has proper maintenance 
performed and kept in good working order.
4.  Reviews and evaluates new products, equipment.
5.  Evaluates and implements new procedures as necessary.
6.  Makes supply decisions with sufficient accuracy so no supplies run out 
or remains beyond the expiration date.
7.  Responsible for daily quality control and quality assurance to include 
establishing training and maintaining Q.C. and Q.A  protocol with subordinate 
staff.
8.  Coordinates monthly QA and QC data.
9.  Ensures that laboratory section meets all safety and other regulatory 
requirements.
10.  Effectively manages technical aspects of hospital accounts
11.  Gathers information and carries through to completion assigned projects 
while adhering to established time frames.
12.  Prepares periodic reports as necessary.
13.  Assures activities of assigned section are coordinated to insure quality 
patient care and economics of operation.
14.  Participates in continuing education as required.
15.  Consults with LIS Administrator to troubleshoot LIS problems and 
enhancements.
16.  Participates in the development of assigned section and department wide 
policies and procedures.
17.  Maintains all section records relating to operation such as quality 
control and productivity statistics, incident reports and the like.
18.  Reviews and summarizes records and prepares reports for management review.
19.  Develops and maintains cooperative working relationships with physicians 
and various other employees.
20.  Provides advice and direction to subordinates for technical problems 
encountered. Explains and demonstrates appropriate techniques or methods as 
necessary.
21.  Performs routine procedures in achieving workload demands.
22.  Maintains knowledge of current trends and developments in the field of 
expertise.
23.  Performs other duties as assigned

POSITION REQUIREMENTS:

Education and formal training:
Knowledge

Re: [Histonet] IHC theoretical question

[Adam .]

Hi,

I find this confusing myself sometimes, so I'll try my best to explain this.
I stain mouse tissues. Let's say that I want to stain for the classic
macrophage F4/80 marker, so I go buy a rat anti-mouse F4/80. In order to
further amplify, I buy a biotinylated donkey anti-rat antibody, and then
detect that with HRP/DAB.

Certain parts of the section may be sticky and nonspecifically bind proteins
or even have an affinity for antibodies. If I didn't block, my primary and
secondary antibodies might just bind to them, adding a lot of background.
Pre-incubating with protein such as serum, BSA, or milk will bind to those
and prevent nonspecific binding of your primary or secondary antibodies.
Serum is typically used because it contains antibodies, so you would also
saturate anything that is nonspecifically but preferentially binding
antibodies such as Fc receptors (although sometimes you have add Fc block if
this is particularly problematic).

Typically serum from the secondary antibody host (in this case, donkey) is
used. Let's say your tissue has sticky parts and instead of blocking with
donkey serum as you should, you accidentally block with rat serum. Now your
entire section is coated with low levels of rat IgG that was in your rat
serum, so when you add your anti-rat secondary antibody, it will bind
everywhere. If you block with rabbit serum, there still is a chance that
your anti-rat secondary may cross react with the rabbit IgG. Now if you use
donkey serum, there is pretty much no chance that the donkey raised
antibodies against donkey IgG unless the donkey had some bizarre autoimmune
disease. So that's why you typically add serum derived from the secondary
antibody host.

Hopefully, your antigen doesn't nonspecifically bind stuff, or else IHC is
going to be very hard. Most antigens don't nonspecifically bind stuff, so
you're good.

Is that clear?
Adam

On Fri, Dec 24, 2010 at 10:30 AM, wassan alkadhumi w_alkadh...@yahoo.comwrote:

 Dear histonet members
 I have a theoretical question concerning IHC, we do HRP method using DAKO
 materials, first step in immuon staining is to add peroxidase blocking
 solution
 to quench endogenus peroxidase. The second step is what am having problem
 with,
 we prepare solution from human serum diluted in wash buffer 1:2, as my
 understanding to why we do this step is to block unwanted proteins in the
 tissue
 so that we prevent background staining, but this step is done before adding
 primary antibody, wont this step block the antigen too since the antigen
 is a
 protein too?
 the other theory i hired is the proteins tend to coagulate together so we
 add
 this solution to dispense them, primary antibody then can easily attach to
 the
 antigen.
 we have two histotext books in the hospital and i red the IHC
 sections, their
 was really nothing clear about why we do this step and i searched the
 Internet
 with no satisfactory answer.
 As u can see i need help!
 step #3 primary Ab
 step#4 link system(secondary Ab against primary Ab (protein)
 Step #5 chromogen DAB
 Am doing IHC for a year and 8 months, till now am confused about this step
 and
 when i train people am not sure what to say to them about it.
 help me please
 Have a great holy day

 Wassan
 Histotechnician
 Shorsh hospital
 North of Iraq



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[Histonet] Hazy background during IF

[Adam .]

Seasons Greeting from the laboratory:

I've been having a somewhat puzzling issue with my IFs lately. I stain
mostly fixed, decalcified mouse bones, and recently, my sections have this
strange haze over them. It's not consistently but it does crop up in maybe
10% of sections. It looks like a dense fog settled over my sections,
especially in the red/594/TxRed channel, often obscuring the signal. It
sometimes does this it the green channel and rarely in the DAPI channel. If
I carefully strip off the coverslip and soak the sections in PBS for a few
minutes, and then re-mount, the problem goes away. I'm guessing it's a
problem in the mounting somewhere.

Here is my protocol:
Sections are circled with a PAP pen. All liquids are dispensed using a 200
uL pipetter onto it
Finish staining
Wash 3 x 5' in TBS-T
Add a drop of Prolong Gold Antifade (I previously used Vectashield and had
the same problem)
Put on coverslip
Dab corners of coverslip with clear nailpolish
Let sit overnight for mounting media to cure

Any ideas what could be going on?

Thanks,
Adam
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Re: [Histonet] Question from one of our researchers

[Adam .]

While not a cell membrane marker, you can often use differential
interference contrast (DIC) microscopy. The microscope I use has a special
setting that lets you add DIC without any additional staining. In the mouse
bone marrow paraffin sections, it lets you see most edges of most cells.

Adam

On Wed, Dec 1, 2010 at 11:19 AM, Johnson, Teri t...@stowers.org wrote:

 Can someone give me ideas to pass along to one of our researchers? Of
 course adhesion molecule antibodies are the first thought, but not for
 heterogeneous cell populations. So I was wondering maybe an antibody
 cocktail? Are there any lectins that might show this? Thanks!

 I am wondering what kind of cell membrane marker is recommended to stain
 tissue section for imaging (going to process the image with 3D Imaris
 software).
 Cells are mouse tissue section in paraffin and very heterogeneous. To see
 cell-cell boundary clearly.

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[Histonet] Paraffin liver sections

[Adam .]

Dear all,

One of my colleagues has consulted me about some paraffin embedded mouse
livers he's been trying to cut. They were fixed in neutral buffered formalin
for 3 days and then processed and embedded into paraffin. He was trying to
cut 5 uM sections on our microtome, except as soon as the blade hit the
liver, the liver would start to roll up and collapse into a small sliver,
and you'd just get sections of paraffin with a hole where the liver should
be. We tried changing cutting angle and soaking the blocks in ice water with
no success. This is the first time we've tried livers, but we routinely cut
decalcified bones on that microtome and have never seen that. Any ideas on
what is going on?

Thanks,
Adam
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Re: [Histonet] Paraffin liver sections

[Adam .]

Thanks to all the prompt responses. He fixed the entire liver in excess
formalin (I think around 15 mL) with rocking for 3 days. I'm not sure about
the processing schedule; I'll have to contact the histology core to ask.

I forwarded all of the advice along, and he tried soaking the blocks in ice
water for several minutes. It fixed most of the problem, at least for now.
You people are far too helpful.

Thanks,
Adam

On Thu, Nov 4, 2010 at 2:53 PM, Jay Lundgren jaylundg...@gmail.com wrote:

 Sounds to me like they are overprocessed (dried out).  Try soaking them on
 ice for 30 minutes or so before cutting.  Some people use a drop of fabric
 softener on their ice tray to soften the tissue, or there are commercial
 products like soft block from Polyscience.

  Jay A. Lundgren M.S.,
 HTL (ASCP)










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[Histonet] (no subject)

[Adam Hacking]

Hi,
 
Does anyone have a blade holder, tungesten blade and clamp for holding 
specimens that would fit on a Leica 2265 ? I'm trying to upgrade a microtome 
set up for paraffin sectioning to one that can handle resin embedded sections.
 
Many thanks
 
Adam



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[Histonet] Microtome for undecalcified tissues

[Adam Hacking]

Hi,
 
I'm looking for a good used sledge or rotary microtome capable of cutting MMA 
emebdded bone specimens. Can anyone reccomend a good model, manufacturer, 
supplier or know of a good used machine ?
 
Many thanks.
 
Adam



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[Histonet] RE: Cassette Marking

[Adam Harris]

Hi All,

If anyone would like to try a free sample of the KP Lab marker for
cassette, slide, or general purpose labeling, feel free to contact me and I
will be more than happy to send you one. It's a win-win situation!! 

Adam Harris
Sales Associate 
Sensor Health Inc. 
110-6 Turnbull Crt.
Cambridge, ON
N1T 1K6
T: 1-888-777-7080
T: 519-621-1515
F: 519-621-8778
www.sensorhealth.com

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
histonet-requ...@lists.utsouthwestern.edu
Sent: None
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 83, Issue 33

Send Histonet mailing list submissions to
histonet@lists.utsouthwestern.edu

To subscribe or unsubscribe via the World Wide Web, visit
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
or, via email, send a message with subject or body 'help' to
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When replying, please edit your Subject line so it is more specific
than Re: Contents of Histonet digest...


Today's Topics:

   1. RE: Cassette Marking (hymclab)
   2. Autofluorescence and literature for getting rid of the
  problem  (gayle callis)
   3. Re: Cassette Marking (Sean McBride)
   4. Re: Cassette Marking (Victor Tobias)
   5. RE: Cassette Marking (sgoe...@xbiotech.com)
   6. RE: Cassette Marking (Vanessa Avalos)
   7. RE: Autofluorescence and literature for getting rid ofthe
  problem (WILLIAM DESALVO)
   8. RE: Cassette Marking (Sherwood, Margaret )
   9. Re: Cassette Marking (histot...@imagesbyhopper.com)
  10. Coverslipping video (Caroline Bass)
  11. RE: Plain Vanilla Autostainer? (susan.wal...@hcahealthcare.com)
  12. RE: Cassette Marking (Nita Searcy)
  13. m.bovis (Kathleen Jones)
  14. RE: Plain Vanilla Autostainer? (Cheri Miller)
  15. TN  -   HT Opprotunity  (Hale, Meredith)
  16. RE: RE: Plain Vanilla Autostainer? (Sherwood, Margaret )
  17. RE: RE: Plain Vanilla Autostainer? (Bernice Frederick)
  18. Laser Capture Machine (Reuel Cornelia)
  19. Histotech needed can you help? (Pam Barker)
  20. Job Opening in San Diego (Alyssa Peterson)


--

Message: 1
Date: Tue, 19 Oct 2010 14:49:18 -0500
From: hymclab hymclab.hymc...@ministryhealth.org
Subject: RE: [Histonet] Cassette Marking
To: 'Sherwood, Margaret ' msherw...@partners.org, Nita Searcy
nsea...@swmail.sw.org, histonet@lists.utsouthwestern.edu
histonet@lists.utsouthwestern.edu
Message-ID:

ef3001233998854390c692d19b058f0f6d8a650...@exmhcmbx01vs.ministryhealth.net

Content-Type: text/plain; charset=us-ascii

We use the pens from Statlab also and love them.

Dawn D. Schneider, HT(ASCP)
Lead Histology Tech
Howard Young Medical Center
240 Maple St.
Woodruff, WI  54568
715-356-8174


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Sherwood,
Margaret
Sent: Tuesday, October 19, 2010 1:38 PM
To: Nita Searcy; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Cassette Marking

The marking pencils work fine and we just ordered some marking pens from
StatLab which work great.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nita Searcy
Sent: Tuesday, October 19, 2010 2:22 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cassette Marking
Importance: High

** High Priority **

If you HAVE to manually mark cassettes - what are you using? Cassette pens ?
Pencils ? What is the rest of the world doing?

Anything else on the market?

Thanks



Nita Searcy, HT/HTL (ASCP)
Scott and White Hospital
Division Manager, Anatomic Pathology
2401 S. 31st. Street
254-724-2438
Temple, Texas, 76502
nsea...@swmail.sw.org


254-724-2438



The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the
e-mail
contains patient information, please contact the Partners Compliance
HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you in
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but does not contain patient information, please contact the sender and
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it or its contents

Re: [Histonet] chicken EGFP aby

[Adam .]

I've had good luck using Abcam's chicken polyclonal:
http://www.abcam.com/GFP-antibody-ab13970.html

I use a directly conjugated secondary, and the staining is usually pretty
bright. But then again, my GFP expression is high.

Adam

On Thu, Oct 7, 2010 at 7:44 AM, Susan Travers traver...@osu.edu wrote:

 Does anyone have experience with detecting EGFP with some of the current
 aby's made in chicken?  We have used aby's from both AVES labs and Millipore
 with negative results. In both cases we used the biotinylated secondary aby
 from AVES followed by a fluorescent streptavidin.  Absolutely no staining.

 Using the same tissue, we were able to use a different aby made in rabbit
 and it worked great. However, because of double-labeling needs we'd really
 like to get the chicken to work.

 Perhaps someone has experience or insights?

 Thanks!
 Susan Travers
 Division of Oral Biology
 College of Dentistry
 The Ohio State University

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Re: [Histonet] Bone IHC

[Adam .]

I've been experimenting with different ways to solve this problem myself. I
fix my tissues in 10% zinc buffered formalin and decalcify in formic acid
for 72 hours, followed by embedding and cutting 5 uM sections.

From trial and error, I've determined that incubating the slides flat on a
slide warmer at 37C (or in my case, the bottom of an unused bacterial
incubator) can prevent nearly all the detachment you observe but it's time
dependent. I think the problem is that the sections get small amounts of
water underneath them when you scoop them up off the water surface during
sectioning and during HIER, that water boils and shears off the slide.

If you leave the slides overnight, the slides were get destroyed during
HIER. However, if you leave them for a week, they tend to be nearly
untouched even at 95C for 10 mins. I'm currently in the process of
determining if a few days is enough time.

Adam

On Thu, Oct 7, 2010 at 2:20 PM, Liz Chlipala l...@premierlab.com wrote:

 We lower the temp of retrieval to 70C for 2 hours and have good success
 with that.

 Liz

 Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
 Manager
 Premier Laboratory, LLC
 PO Box 18592
 Boulder, Colorado 80308
 office (303) 682-3949
 fax (303) 682-9060
 www.premierlab.com


 Ship to Address:
 1567 Skyway Drive, Unit E
 Longmont, Colorado 80504

 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu
 [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Vanessa
 J. Phelan
 Sent: Thursday, October 07, 2010 1:14 PM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Bone IHC

 Hi everyone,

 I was wondering if anyone has any tricks on how to get bone sections to
 stop lifting off the slide through the IHC process? I leave them in the
 oven for quite a while to make sure they are baked on, however after
 antigen retrieval  (pressure cooker for 20mins) most of the boney part
 of the tissue comes off and the marrow and muscle stays put! The
 sections are cut onto superfrost plus slides.

 Any help would be much appreciated, thanks.

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Re: [Histonet] Tyramide

[Adam .]

I've had good luck with the biotinyl tyramide system from Perkin-Elmer

Here is what I do using an unlabeled primary antibody
1) Block endogenous peroxide since you use peroxidase chemistry for this to
work
2) Block avidin
3) Block biotin
4) Block with TNB buffer (the kit's equivalent of blocking serum)
5) Add primary antibody
6) Add biotinylated secondary antibody
7) Add SA-HRP (comes with kit)
8) Add biotinyl tyramide reagent. The HRP will deposit biotinylated tyramide
near your antigen.
9) Add SA-HRP
10) Add DAB

It works well. It also works if you replace step 9 with SA-fluorophore and
don't use DAB at all.

If your primary antibody is already labeled with HRP, you could do try this:

1) Block endogenous peroxide since you use peroxidase chemistry for this to
work
2) Block avidin
3) Block biotin
4) Block with TNB buffer (the kit's equivalent of blocking serum)
5) Add primary HRP conjugated antibody
6) Add biotinyl tyramide reagent
7) Add SA-HRP
8) Add DAB

I've never tried it, but I'd expect it won't amplify as well as my method.
However, I see no reason why it shouldn't work.

Good luck,
Adam

On Tue, Sep 28, 2010 at 11:13 AM, sgoe...@xbiotech.com wrote:


   Hello  world...So  trying  to  amplify my signal using tyramide.  ; I
   bought  a  kit  from  Invitrogen  to  use  with  fluorescence  and the
   fluoresce  nce didn't work at all!  The PHD that is helping with this
   said  that  h  e  thought  we  should try it using DAB instead of the
   fluor.   I  called  t  he  Invitrogen technical help...WOW!!!  I have
   never  had  my  own questio n repeated back to me so many times, that
   guy  was  a  moron on IHC, rude, and should be fired!!!  So I come to
   ya'll!!   Any  idea  where  I can b uy tyramide to be used with a DAB
   developer?   My  primary  antibody is a lready conjugated with HRP, I
   just  want  to  amplify  with  with  the  tyramide  a  nd  stain with
   DAB/hematoxylin.  Any help would be awesome!!!


   Sarah Goebel, B.A., HT (ASCP)



   Histotechnician

iX Biotech USA Inc.

   8201 East Riverside Dr. Bldg 4 Suite 100

   Austin, Texas nbsp; 78744

   (512)386-5107
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Re: [Histonet] Ventana Renaissance Containers

[Adam Harris]

Hi Ricky,

We may have what you need, we have multiple sizes of containers which we can
either send empty or can fill for you whichever you prefer. Check out the
site if you would like at www.sensorhealth.com 

Hope that helps.

Adam Harris
Sales Associate 
Sensor Health Inc. 
110-6 Turnbull Crt.
Cambridge, ON
N1T 1K6
T: 1-888-777-7080
T: 519-621-1515
F: 519-621-8778
www.sensorhealth.com
-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
histonet-requ...@lists.utsouthwestern.edu
Sent: None
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 81, Issue 23

Send Histonet mailing list submissions to
histonet@lists.utsouthwestern.edu

To subscribe or unsubscribe via the World Wide Web, visit
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
or, via email, send a message with subject or body 'help' to
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When replying, please edit your Subject line so it is more specific
than Re: Contents of Histonet digest...


Today's Topics:

   1. RE: CD34 positive control (McMahon, Loralee A)
   2. Ventana Renaissance Containers (ricky hachy)
   3. CD34 Control (Silverman, Jeffrey)


--

Message: 1
Date: Wed, 18 Aug 2010 12:17:39 -0400
From: McMahon, Loralee A loralee_mcma...@urmc.rochester.edu
Subject: RE: [Histonet] CD34 positive control
To: Joel Reichensperger jreichensper...@siumed.edu, Histonet
histonet@lists.utsouthwestern.edu
Message-ID:

c27aa2a01cef31469813089e226f582e02d5a7c...@urmcms7.urmc-sh.rochester.edu

Content-Type: text/plain; charset=us-ascii

Just about any tissue should work.   It stains for Hematopoietic
stem/progenitor cells, bone marrow stromal cells, endothelial cells,
embryonic fibroblasts 
We use a sausage style control here with about 30 different types of tissue.



Loralee McMahon, HTL (ASCP)
Immunohistochemistry Supervisor
Strong Memorial Hospital
Department of Surgical Pathology
(585) 275-7210

From: histonet-boun...@lists.utsouthwestern.edu
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joel Reichensperger
[jreichensper...@siumed.edu]
Sent: Wednesday, August 18, 2010 12:05 PM
To: Histonet
Subject: [Histonet] CD34 positive control

  I have a doctor who wants to stain some tissue with cd34. I need to
know if anyone can recommend a good positive control tissue for this
antibody. The staining will be done in paraffin embedded sections.
Thanks in advance.

Joel

--
Joel Reichensperger
Researcher II
Southern Illinois University
Plastic Surgery Institute
jreichensper...@siumed.edu
217-545-7309 (Office)
217-545-1824 (Fax)


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--

Message: 2
Date: Wed, 18 Aug 2010 16:28:39 +
From: ricky hachy elc...@hotmail.com
Subject: [Histonet] Ventana Renaissance Containers
To: Histonet histonet@lists.utsouthwestern.edu
Message-ID: snt114-w54ab5f08e7e0cfc97193edcf...@phx.gbl
Content-Type: text/plain; charset=iso-8859-1


Hello ,
 
I need 4 good plastic containers, where you fill with xilene,alcool...
for the VENTANA RENAISSANCE . 
 
Anyone could sell me ?
 
Regards
 
Ricky 

--

Message: 3
Date: Wed, 18 Aug 2010 12:30:58 -0400
From: Silverman, Jeffrey jsilver...@nshs.edu
Subject: [Histonet] CD34 Control
To: 'jreichensper...@siumed.edu' jreichensper...@siumed.edu
Cc: 'histonet@lists.utsouthwestern.edu'
histonet@lists.utsouthwestern.edu
Message-ID:
83f4d81747a7094dafe3ae87151ecb941ce4144...@sykechxvs01.nslijhs.net
Content-Type: text/plain; charset=us-ascii

Joel,
A section of skin will be a fine CD34 control. All collagenous connective
tissue, including the dermis,  are rich in CD34+ dendritic interstitial
fibroblasts as well as CD34 positive endothelium in the resident vessels.
Actually, in my lab we use a section of fallopian tube from tubal ligation,
they are loaded with the fibroblasts and vessels.

Jeff Silverman



--

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End of Histonet Digest, Vol 81, Issue 23



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[Histonet] Microscope Slide stamp

[Adam Harris]

Hi Jill,

 

I know this was a while ago but if you are still in need of
something to help label your slides we are carrying an Ultra Fine Lab Marker
that works really well on the frosted ends of slides. It is resistant to
alcohol and xylene, and writes very nicely on smaller areas. Feel free to
check us out at www.sensorhealth.com http://www.sensorhealth.com/  for
more details and let us know if you would like to try a free sample. 

 

 

Adam Harris

Sales Associate 

Sensor Health Inc. 

110-6 Turnbull Crt.

Cambridge, ON

N1T 1K6

T: 1-888-777-7080

T: 519-621-1515

F: 519-621-8778

www.sensorhealth.com

 

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Re: [Histonet] IHC

[Adam .]

I always leave my primary antibodies on overnight at 4C. I've seen some
protocols for tricky antigens where people leave it on for days. I wouldn't
leave it a room temperature though.

Go home and enjoy your Friday,
Adam

On Fri, May 14, 2010 at 4:37 PM, sgoe...@xbiotech.com wrote:


   Can  you  leave  your  primary antibody on slides overnight (or the w
 eekend)  or  will  this  screw  things  up?   I  don't want to be here
   another couple of hours for secondary, etc. on a Friday!!


   Sarah Goebel, B.A., HT (ASCP)

iH istotechnician

   XBiotech USA Inc.

   8201 East Riverside Dr. Bldg 4 Suit e 100

   Austin, Texas  78744

   (512)386-5107
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Re: [Histonet] IF Doublestaining

[Adam .]

In my experience, most fluorophores are quite stable to many washes. Here is
what I would try at first:

1) Stain with rat anti-mouse and rabbit anti-human overnight at 4C in a
humidified chamber. Dilute each antibody in a single volume of your favorite
staining buffer (PBS, TBS, TBS-T). For example, if your rat antibody needs
to be diluted in 1:100 and your rabbit needs to be diluted 1:200, take an
aliquot of 200 uL of buffer and add 2 uL of the rat primary and 1 uL of the
rabbit. Add the appropriate volume (usually 50-200 uL) of that mixture to
each slide.

2) Wash the next day, and add your secondaries again to a single mixture for
1 hour at room temperature. As long as your secondaries have been highly
cross adsorbed to many species, you shouldn't have a problem.

3) Wash, counterstain, mount, enjoy.

I really don't think you need to do sequential staining for this. I've heard
anecdotal reports of two primaries or two secondaries forming immune
complexes when mixed together, but I've never really had that problem.

Good luck,
Adam

On Thu, May 13, 2010 at 1:00 PM, Igor Deyneko igor.deyn...@gmail.comwrote:

 Hello Everyone!
 I'm planning to try some IF co-staining with 2 antibodies, one is a
 rat-anti-mouse and the other one is rabbit anti-human on a  xenograft, each
 has an appropriate secondary, donkey anti rat and donkey anti-rabbit,
 conjugated to 488 and 593. Can someone advise the best way to perform such
 a
 procedure, I'm afraid the rules of sequential staining might not work due
 to
 fluorophore instability with washes. if anyone has performed such type of
 stain, i would appreciate any tips.
 Thank you.
 Igor Deyneko
 Infinity Pharmaceuticals
 Cambridge, MA 02139
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Re: [Histonet] list responses

[Adam .]

Rather than doing a simple reply, choose reply all.

Adam

On Wed, Apr 14, 2010 at 2:28 PM, Carrie Disbrow dis...@shands.ufl.eduwrote:

 Hi
 We kept wondering why our responses to the discussion are not seen on the
 daily list. We then realized the response must be going directly to the
 person who wrote the original post.  Is there anyway the responses can be
 seen on the list as we are losing the input of the others.
 Thanks,
 Carrie

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[Histonet] Troubleshooting IHC

[Adam .]

Hi all,

I've recently run into a problem troubleshooting IHC on mouse bones. I am
using the tyramide amplification system using a goat primary, anti-goat
biotin, strepavidin-HRP, biotinyl tyramide, followed by another
strepavidin-HRP and then DAB+ from Dako. When I use an isotype goat IgG, I
get essentially no staining.

I titered it once for immunofluorescence (SA-fluor instead of HRP in the
last step) and got a titer of 1:800, and these staining conditions are quite
reproducible. Then I titered for IHC, and 1:200 gave the best signal to
noise. I did a batch of staining using those IHC conditions, and it stained
beautifully, comparable to my IF. I then tried to repeat it on a second
batch of sections processed in the same way, and the background was terrible
(but not on my isotype slide). I thought maybe it was a problem in
processing so I did a third batch of sections, and the background was still
really bad. I see real staining sometimes, but I need to quantify this
staining using histomorphometry, so I really need clean staining. Any ideas?
The only thing I can think of is that 1:200 is just at the limit of
titration that gives too much background.

Thanks,
Adam
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[Histonet] Re: Troubleshooting IHC

[Adam .]

Yes. I block in 3% H2O2, followed by protein block (it's a mysterious buffer
called TNB that comes with the tyramide amplification kit), and then
avidin/biotin.

Adam

On Thu, Mar 25, 2010 at 2:25 PM, Margaryan, Naira 
nmargar...@childrensmemorial.org wrote:

  Hi Adam,



 How do you block?



 I usually have: H2O2, Avidin/Biotin and Protein blocking steps.



 Naira



 Message: 12

 Date: Thu, 25 Mar 2010 10:43:28 -0500

 From: Adam . anonwu...@gmail.com

 Subject: [Histonet] Troubleshooting IHC

 To: histonet@lists.utsouthwestern.edu

 Message-ID:

   858249121003250843h2d0f16a2q1a74ad1091630...@mail.gmail.com

 Content-Type: text/plain; charset=ISO-8859-1



 Hi all,



 I've recently run into a problem troubleshooting IHC on mouse bones. I am

 using the tyramide amplification system using a goat primary, anti-goat

 biotin, strepavidin-HRP, biotinyl tyramide, followed by another

 strepavidin-HRP and then DAB+ from Dako. When I use an isotype goat IgG, I

 get essentially no staining.



 I titered it once for immunofluorescence (SA-fluor instead of HRP in the

 last step) and got a titer of 1:800, and these staining conditions are
 quite

 reproducible. Then I titered for IHC, and 1:200 gave the best signal to

 noise. I did a batch of staining using those IHC conditions, and it stained

 beautifully, comparable to my IF. I then tried to repeat it on a second

 batch of sections processed in the same way, and the background was
 terrible

 (but not on my isotype slide). I thought maybe it was a problem in

 processing so I did a third batch of sections, and the background was still

 really bad. I see real staining sometimes, but I need to quantify this

 staining using histomorphometry, so I really need clean staining. Any
 ideas?

 The only thing I can think of is that 1:200 is just at the limit of

 titration that gives too much background.



 Thanks,

 Adam



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Re: [Histonet] Anti-human abs work on porcine tissue

[Adam .]

I routine use anti-human antibodies to stain mouse tissue, so it can be
done. It completely depends on the antigen they used to immunize. If the
human and pig proteins are highly homologous or identical, then it has a
good chance of working. If they're not, you're in uncharted territories. If
you ask nicely, most companies will tell you what antigen or antigen
fragment they used, but most don't actively advertise this.

Adam

On Wed, Mar 17, 2010 at 8:15 PM, RICKY MATHIS cmmath...@bellsouth.netwrote:

 I work with some porcine tissues and it is sometimes difficult to find
 antibodies specific to pig.  A doctor I work with asked some one he knew in
 Europe about using antibodies that are listed as working in human being used
 on porcine tissue.  This person stated that there would be about a 10%
 chance that the anti-human antibodies would work on porcine tissues.   The
 antibody companies mostly give the same we did not try it on pig tissue
 response.  I understand that it is likely expensive to continue to test
 antibodies on a wide variety of animal tissues, so that is fine.  But what
 do you guys think about the 10% chance?  I would have said more than that.
 Thank you in advance for your time,
 Cathy
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Re: [Histonet] Unsubscribe please

[Adam .]

I think a lot of grief could be saved if the managers of HistoNet added the
following four words at the bottom of each e-mail. Who would I need to
contact to make such a suggestion?

http://lists.utsouthwestern.edu/mailman/listinfo/histonet to change
subscription preferences

Adam

On Tue, Mar 16, 2010 at 10:08 AM, Emily Sours talulahg...@gmail.com wrote:

 JUPITER'S THUNDER!
 GO TO THIS WEBSITE.
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet
 SCROLL DOWN TO THE BOX WHERE IT SAYS UNSUBSCRIBE (MAKE SURE YOU USE
 THE CORRECT BOX OR YOU WILL NOT BE UNSUBSCRIBED) AND TYPE IN YOUR
 EMAIL.
 CLICK ON THE BOX THAT SAYS UNSUBSCRIBE.
 DO NOT WRITE TO THE LIST.

 A state-imposed metaphysic or religion should be opposed, if necessary
 at pistol-point.  We must fight for variety if we fight at all.  The
 uniform is as dull as a sculptured egg.
 --Lawrence Durrell, Consequential Data, Balthazar



 On Tue, Mar 16, 2010 at 10:57 AM, Barbara Ratner b...@merraine.com wrote:
 
 
  Barbara Ratner
  Vice President
  Merraine Group Inc.
  One Executive Blvd.
  Suite #110
  Montebello, NY 10901
 
  direct: (845) 290-1900
  main: (845) 357-3355 x104
  fax (212) 918-9184
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  www.merraine.com http://www.merraine.com/
 
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Re: [Histonet] Double labeling with antibodies that need different fixatives

[Adam .]

Although I haven't tried it myself, others have gotten CD31 from BD to work
on FFPE tissue using the tyramide amplification system on zinc buffered
formalin fixed sections. I've generally had good luck with zinc buffered
formalin myself for many antigens so it may work for your other one.

See http://www.ncbi.nlm.nih.gov/pubmed/19052548

Just to be clear, they used zinc buffered formalin, which isn't the same
thing as zinc fixative.

Adam

On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe 
pverbrug...@meddent.uwa.edu.au wrote:


 Hi all,

 I would like to do an immunofluorescent double labeling with two
 antibodies but 1 antibody works on acetone fixed frozen tissue but not
 on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370)
 and the other one works on formalin fixed paraffin embedded tissue but
 not on acetone fixed frozen tissue. Is there any way I could still do a
 double labeling and how?
 Also, does anyone have experience with zinc fixative? If my antibody
 works on formalin fixed tissue is it likely to also work on zinc fixed
 tissue?

 Thank you very much in advance,

 Phebe

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Re: [Histonet] Double labeling with antibodies that need different fixatives

[Adam .]

I have no idea if it would work with regular formalin. In my experience,
many antigens work as well in zinc buffered formalin without any antigen
retrieval as regular formalin with antigen retrieval. But really, you just
have to try it yourself.

On antibodies I've gotten this to work, I use commercially available zinc
buffered formalin which comes in gallon jugs for around $50. We don't do any
special processing. We plop our sample (bones) in zinc formalin overnight at
4C, decalcify in EDTA or formic acid (only necessary for bones), and then
embed just like any other sample.

Adam

On Mon, Feb 22, 2010 at 9:45 PM, Phebe Verbrugghe 
pverbrug...@meddent.uwa.edu.au wrote:

  Hello Adam,

 Thank you very much for this very useful information!
 Do you know whether this would also work on tissue fixed with formalin
 instead of zinc buffered formalin by any chance?
 Also, could you give me the recipe for the zinc formalin and can I use a
 standard tissue processor for embedding in paraffin or should I use a
 specific protocol manually and if so, which?

 Thanks!

 Phebe

  --
 *From:* Adam . [mailto:anonwu...@gmail.com]
 *Sent:* Tuesday, 23 February 2010 10:52 AM
 *To:* Phebe Verbrugghe
 *Cc:* histonet@lists.utsouthwestern.edu
 *Subject:* Re: [Histonet] Double labeling with antibodies that need
 different fixatives

 Although I haven't tried it myself, others have gotten CD31 from BD to work
 on FFPE tissue using the tyramide amplification system on zinc buffered
 formalin fixed sections. I've generally had good luck with zinc buffered
 formalin myself for many antigens so it may work for your other one.

 See http://www.ncbi.nlm.nih.gov/pubmed/19052548

 Just to be clear, they used zinc buffered formalin, which isn't the same
 thing as zinc fixative.

 Adam

 On Mon, Feb 22, 2010 at 8:41 PM, Phebe Verbrugghe 
 pverbrug...@meddent.uwa.edu.au wrote:


 Hi all,

 I would like to do an immunofluorescent double labeling with two
 antibodies but 1 antibody works on acetone fixed frozen tissue but not
 on formalin fixed paraffin embedded tissue (CD31 BD pharmingen 553370)
 and the other one works on formalin fixed paraffin embedded tissue but
 not on acetone fixed frozen tissue. Is there any way I could still do a
 double labeling and how?
 Also, does anyone have experience with zinc fixative? If my antibody
 works on formalin fixed tissue is it likely to also work on zinc fixed
 tissue?

 Thank you very much in advance,

 Phebe

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Re: [Histonet] IF staining on peritoneal macrophages

[Adam .]

When I did it, I think I used our standard sort buffer, which is 0.2% BSA in
PBS pH 7.4. I've also read that you can use anything with serum, typically
5-10%. The key seems to be prewetting the membranes with something with
protein.

I usually cytospin them and briefly (30 seconds) air dry them until there is
no obvious liquid, before I added the fixative. I managed to get usable (but
not ideal) cytospins from as few as 1000 cells.

Let me know if it works,
Adam

On Fri, Feb 19, 2010 at 9:47 AM, Mauricio Avigdor
bitesizell...@gmail.comwrote:

 Thank you Adam and Jay for your replies.

 Cytoslides are the pre-marked slides for use with the Cytospin -
 http://www.thermo.com/com/cda/product/detail/0,1055,21035,00.html

 Adam, do you have a recommended concentration of BSA for this? Also, do you
 air dry the slides prior to fixation? Do you air dry afterwards? Thanks
 again for your help.

 Mauricio
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Re: [Histonet] IF staining on peritoneal macrophages

[Adam .]

The best way I know to get cells to stick is to first cytospin liquid with
some protein (BSA or serum) onto the filters to pre-wet them. After that's
done, cytospin your cells. I've gotten this to work with fairly rare FACS
sorted cells.

I've also found that the smaller volume the cells are in, the better your
cytospin. Ideally is 100 - 200 uL. For FACS sorted cells, this means you
want to sort into as small a volume as possible. For lavage cells, that may
mean spinning them down first, although for FACS cells, the cells are
fragile and don't like being centrifuged.

I've gotten cells to stick to the slides by circling the cells with a PAP
pen and then just putting fixative inside PAP while the slides are lying
horizontally.

Adam

On Thu, Feb 18, 2010 at 5:00 PM, Mauricio Avigdor
bitesizell...@gmail.comwrote:

 Greetings all,

 I am trying to do immunofluorescence on peritoneal macrophages. I am having
 a couple of issues that I was hoping one of you could help me resolve.

 Firstly, I am having uneven results with the Cytospin. Cells tend to get
 washed off the slides during rinses. Does anyone have tips on how to make
 cells stick a little better to Cytoslides? Secondly, I have not yet found a
 satisfactory method for fixation. In order to prevent loss of cells when
 dipping the slides into fixatives, I am having to air dry the slides before
 I can do anything to them. I tried the Shandon Collection Fluid (ethanol,
 isopropanol, carbowax) with great success, but it killed the fluorescence.

 I am leaning towards spinning the lavage fluid until I get a pellet and
 then
 resuspending in PBS with a little BSA added. I hope this makes cells stick
 well enough that I can put the slides in formalin or acetone.

 Any thoughts are appreciated!
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Re: [Histonet] Alternatives to BioQuant for Bone Histomorphometry

[Adam .]

I am planning on using IHC using DAB stain to label the cells of interest (a
subpopulation of bone cells) counterstained with hematoxylin. My coworker
wants to do similar things with TRAP stained osteoclasts. The way I was
trained how to do this would be turn off thresholding completely (the guy
said it doesn't work well enough), outline the contours of the bone, and
then outline the perimeter lined by the cells of interest, and finally click
on each cell of interest.

It seemed to me that doing this in BioQuant was needlessly complex and
involved constantly loading and resetting these vectors each time, and if
you forgot to do that, you could do a whole bunch of work and have BioQuant
either compute the parameters completely wrong or simply discard what you
did.

Adam

On Mon, Feb 8, 2010 at 6:45 AM, Jack Ratliff ratliffj...@hotmail.comwrote:

 Adam,

 What stain are you using for your quantitation and are you trying to
 perform measurements via the thresholding feature?


 Jack


 On Feb 5, 2010, at 4:38 PM, Adam . anonwu...@gmail.com wrote:

  Hi all,

 I am looking for an alternative program to BioQuant for bone
 histomorphometry. We need to quantify the number of osteoblasts /
 osteoclasts per bone surface area as well as the percent surface area
 occupied by those cells. We have a computer with BioQuant on it available,
 but we find the software to be incredibly clunky and often nearly
 impossible
 to use. Based on my limited attempts to use it, it very well might rank as
 one of the worst user interfaces I've ever seen, and I was trained in
 computer science and have seen my fair share of horrible software (I'm
 looking at you, Lotus Notes).

 Anyhow, any suggestions on a (preferably cheap / free) replacement for
 doing
 simple analysis or how to make BioQuant less painful would be very
 helpful.

 Thanks,
 Adam
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Re: [Histonet] TRAP Assay and IHC

[Adam .]

I'm by no means an expert for cutting frozen mouse bones, but I've done it.
It's certainly more difficult than other tissues, but you can get decent
sections with lots of practice and patience. When I've gotten it to work,
the bones were fixed in formalin or PFA, decalcified in EDTA, cryoprotected
in 30% sucrose, and then snap frozen in OCT using a beaker of 2-methylbutane
sitting in an ice bucket of liquid nitrogen. The knives we use are nothing
special. I don't post-fix after IHC and usually the sections stick to
Superfrost Plus Slides if you let them dry completely before staining.

I'm not entirely sure what you mean by the tissue lifting out of the
section. Sometimes, the marrow can spill out of the bones or the decalcified
bone can fall off and either pull away from the endosteum or even completely
fall off and splay across the section. More often, the periosteum will pull
away from the OCT a bit. I've found the best way to avoid this is to flatten
the section with a paintbrush the best you can, and then quickly and firmly
plant the slide onto the section. I hold the slide on each between my index
fingers and thumbs and slowly lower it until it almost touches. Then I press
down rapidly so the entire section should get pressed onto the slide as
quickly as possible. If you go slowly and allow the section to pull itself
up onto the slide in increments, you'll often get falling off.

I hope that helps,
Adam

On Thu, Feb 4, 2010 at 9:19 AM, Sherwood, Margaret
msherw...@partners.orgwrote:

 I want to thank everyone who responded to my inquiry re:  TRAP Assay.  I
 don't
 think I made my request that clear re:  IHC.  We want to do both TRAP Assay
 and
 IHC (separately, of course) on mouse tibia.

 We would like to use frozen sections of mouse tibia for IHC.  Our initial
 try to
 cut tibia on the cryostat has resulted in less than optimal sections.  It
 appears that the tissue is lifting out of the section.
 In re:  IHC on frozens -   1)  do you treat the tibia before embedding in
 OCT?
 2) what knife do you cut with on the cryostat? 3)  do you fix slides with
 cold
 acetone before the IHC procedure (we ususally do)?


 Thanks again!

 Peggy


 Peggy Sherwood
 Lab Associate, Photopathology
 Wellman Center for Photomedicine (EDW 214)
 Massachusetts General Hospital
 55 Fruit Street
 Boston, Massachusetts 02114-2696
 617-724-4839 (voice mail)
 617-726-6983 (lab)
 617-726-1206 (fax)
 msherw...@partners.org



 The information in this e-mail is intended only for the person to whom it
 is
 addressed. If you believe this e-mail was sent to you in error and the
 e-mail
 contains patient information, please contact the Partners Compliance
 HelpLine at
 http://www.partners.org/complianceline . If the e-mail was sent to you in
 error
 but does not contain patient information, please contact the sender and
 properly
 dispose of the e-mail.


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[Histonet] Lineage tracing in bone

[Adam .]

Hi all,

As my thesis project moves forward, my commitee has asked me to do some
lineage tracing in mouse bone. For those of you not familiar with this, you
mate a mouse expressing a Cre recombinase to another mouse expressing a
reporter gene with a premature stop codon that can be floxed out. The
reporter is usually lacZ or GFP. The Cre will remove the stop codon and
allow expression of your reporter in the cells the cre is expressed in (and
all its progeny).

Here's the problem: I don't have access to a tape transfer system (yet) so I
have to use fixed decalcified bones. I would prefer to use paraffin embedded
sections due to the improved morphology. To make things more complicated. I
probably will even have to do colocalization with other antibodies.

So here are the options that I am familiar with for reporter strains:

1) lacZ reporters (http://jaxmice.jax.org/strain/003474.html)
I worry that the fixation or embedding will destroy the lacZ. It's pretty
common in developmental biology to see people use these for unfixed tissue
to directly assay lacZ. There was some discussion in the archives (
http://lists.utsouthwestern.edu/mailman/htdig/histonet/2008-February/035626.html)
on a method to embed in paraffin and preserve lacZ but it seems a bit
complicated. Has anyone successfully used an anti-lacZ antibody to do this
in bone?

2) eGFP reporters (http://jaxmice.jax.org/strain/004077.html)
Jackson's website notes that this isn't suitable for immunohistochemistry
because expression is too low. Since bone is so autofluorescent, I really
doubt I'd ever see it.

I do have a chicken anti-GFP antibody, which seems to work really great in
paraffin sections with some mouse strains (col2.3-GFP) but doesn't seem to
work with others (CX3CR1-GFP) when I come in with a Dylight488 anti-chicken.
This seems to occur even though they seem to have similar brightness when
assayed by FACS... I'm not sure why. Has anyone used an anti-GFP antibody
for lineage tracing in bone or other fixed tissues?

If you're aware of any other strains or methods to accomplish this, I would
greatly appreciate your suggestions. There is a small chance that I may get
access to a Cryojane at some point in the future, and I would also welcome
comments on how feasible this would be using that system.

Thanks,
Adam
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Re: [Histonet] 0.1 M PB 0.01M PBS

[Adam .]

I've used 1X PBS for everything and it seems to work.

Adam

On Sun, Jan 17, 2010 at 8:41 PM, TF ti...@foxmail.com wrote:

 We use 0.1 PB for tissue fixation (4% PFA), cryo-preservation (30% sucrose)
 then go 0.01 PBS for IHC

 though these are routines here, I wonder anyone can specify on the use of
 two different solutions, especially why?


 2010-01-18



 TF
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Re: [Histonet] Counterstain for fluorescent tissue

[Adam .]

Hi,

The most common counterstains for fluorescent work is the nuclear
counterstain DAPI, which fluoresces in the UV spectrum. If your
microparticles fluoresce in that wavelength, then you obviously can't use
that but there are a whole host of other nuclear counterstains that
fluoresce in pretty much any part of the spectrum you want (see
http://www.cbit.uchc.edu/microscopy_facility/fluorescent_probes.html for the
most common). Various companies sell antifade mounting media with DAPI
included, which is really easy to use. You section your tissue, add the
mounting media right on top of the section, coverslip, and then seal with a
sealant (usually run-of-the-mill nailpolish).

Now here's the problem. Nuclear counterstains stain nuclei really well, but
unlike hematoxylin, you often can't see the cell boundaries that well.
Sometimes you're lucky in that your tissue is a bit autofluorescent and you
can see the cell boundaries that way, and sometimes you can do fancy
microscope and optical tricks such as differential interference contrast
(DIC). If neither of these work, people use antibodies to highlight some
really abundant protein in your tissue such as cytoskeletal proteins.

For your work, there might even be a simpler solution. If your
microparticles survive hematoxylin staining, you can often just take a
bright field photograph with the blue counterstaining, switch on the
fluorescent filters, take photographs of that, and then merge them together
using image processing software such as Photoshop.

Adam

On Sat, Jan 9, 2010 at 12:46 AM, John Kiernan jkier...@uwo.ca wrote:

 Dear Nick Evans,

 First:  Say who and where you are, and who is in charge of your experiments
 with mice.

 Second:  Tell your boss to buy two or three histotechnology textbooks
 (about $50 each) and allow himself and you and all your colleagues 30 mins
 paid daily reading/lunch time.

 John Kiernan
 Anatomy, UWO
 London, Canada
 = = =
 - Original Message -
 From: Nicholas David Evans ndev...@stanford.edu
 Date: Friday, January 8, 2010 20:18
 Subject: [Histonet] Counterstain for fluorescent tissue
 To: histonet@lists.utsouthwestern.edu

  Dear all,
 
 
 
  I am sorry if this is a bit of a basic question. I would like to
  observelocalization of fluorescent microparticles embedded in
  mouse skin. I plan
  to do this simply by cryosectioning skin, mounting the tissue without
  fixation, and observing under the fluorescence microscope (the
  particlesare added before killing the mouse).
 
 
 
  However, I would also like to counterstain the tissue without
  losing the
  fluorescence of my sample (so I can see similar features as I
  might using
  HE). Can anyone suggest a good way to do this? I would be very
  gratefulfor any advice.
 
 
 
  Many thanks
 
  Nick Evans
 
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Re: [Histonet] mouse Cytokine ICC/IHC protocol help.

[Adam .]

I've managed to get one chemokine (SDF-1) to work on FFPE sections. The key
was using the tyramide amplification system from Perkin Elmer. It's a dirty
staining because it's secreted so you see staining bound up randomly in
noncellular extracellular matrix but you do see it concentrated in some
cells. Since it's not easy, our lab tries to avoid doing this type of
delicate work and instead do ELISAs and intracellular flow cytometry.

Adam

On Mon, Jan 4, 2010 at 12:41 PM, Jamie E Erickson jamie.erick...@abbott.com
 wrote:

 Hi All,  Happy New Year...

 As the new year is upon us I and my fellow histologist feel compelled to
 attempt IHC on mouse tissue again for the detection of various cytokines.
 As I have not had much success in this area in the past I thought I would
 ask if there is anyone out there that you know that may help us on our
 Journey..

 We want to try to detect cytokine (TNF,IL-1,IL-13) along with our soluble
 targets in mouse and rat tissues.
 Ideally we want to use paraffin sections so we will explore
 fixations/processing schedules..and any other voodoo solutions that  might
 work.

 If you or someone you know has a good source of papers/ protocol we might
 download we would love to have them..

 As we have the ability to use mice and rats we will be
 simulating/overexpression of cytokines in-vivo and taking tissues at
 necropsies in various fixations.

 If you can help please let me know.

 Thanks and Happy new Year.

 Jamie Erickson
 Abbott Labs.
 Scientist II, M.S. HTL (ASCP)
 Discovery Safety, Metabolism  Pharmacokinetics
 Phone 508-688-3134
 FAX 508-793-4895
 jamie.erick...@abbott.com
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[Histonet] Directly conjugated secondaries versus biotinylated secondaries

[Adam .]

Greetings,

Hopefully all of you are enjoying a great time with your friends and family
this week rather than working like me. Here is my problem.

I have an antibody (anti-panendothelial antigen, i.e. MECA32 from BD
http://www.bdbiosciences.com/ptProduct.jsp?prodId=19506) and I'm trying to
get it to work on 4% PFA fixed, decalcified, paraffin embedded sections. I
previously titered it to 2 ug / mL with a biotinylated anti-rat F(ab')2 (1
ug / mL) and then a strepavidin Dylight 594 (1 ug / mL) and got beautiful,
strong staining. Those of you who have been helping me from my IHC infancy
would be proud.

However, I really need it to be a directly conjugated secondary because I
want to co-stain with something else that only works with a biotinylated
secondary followed by tyramide amplification. So I retitered it from 5 ug /
mL and down and came in with a Dylight-649 conjugated anti-rat (1 ug / mL)
secondary and I saw nothing (Dylight 649, which is a Cy5 replacement, is
too far red to see without the aid of a camera--although the guy who helped
me use the microscope says there's one woman on campus who has infrared
vision). I should note that another group has reported getting this to work
with a biotinylated primary and adding an fluorophore conjugated avidin.

I can think of a couple of possibilities here
1) The antibody needs that extra edge of a biotinylation to get sufficient
signal (has anyone ever seen this problem which couldn't be solved by
titrating something?)
2) I need to increase the primary titer ever more
3) I need to increase the secondary titer (what primary titer should I use
if I do this?)
4) Dylight 649 is just too dim to see anything and if I switched to another
fluor, it would work. I have no experience with this fluor, as our floor
scope doesn't have the filters for it.
5) This is all a fluke and I screwed something up

Any suggestions on how to troubleshoot this are welcome.

Adam
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Re: [Histonet] RE: Akemi - Urgent!

[Adam .]

Phishing also happens over the phone as well. It is not unheard of that
people get phone calls claiming to be from a bank asking for personal
information (why would a bank need your account number... aren't they your
bank and don't they already have this information?). It is generally a very
bad idea to give any personal information out in any venue other than the
physical office of the institution which you do business. If the company
suspects that your account has been compromised, they will disable it and
then ask you to contact them. If you receive such an e-mail, do not click on
any links in it or dial any phone number contained within. Instead, look up
the company's contact information (in a phone book or via a search engine)
and use the publicly available contact information.

Adam

On Thu, Nov 12, 2009 at 11:04 AM, Rene J Buesa rjbu...@yahoo.com wrote:

 Akemi:
 And that was your second mistake. Why the server, in this case Yahoo, would
 like your password and your birth day when that is not originally needed?
 I NEVER EVER answer anything even if I am told that my life depends on it.
 Never answer a request for information of any kind. If it a legitimate
 request they will call you on the phone.
 René J.

 --- On Thu, 11/12/09, Akemi Allison-Tacha akemiat3...@yahoo.com wrote:


 From: Akemi Allison-Tacha akemiat3...@yahoo.com
 Subject: Re: [Histonet] RE: Akemi - Urgent!
 To: SaraBreeden sbree...@nmda.nmsu.edu, 
 histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu,
 Helen Fedor hfe...@jhmi.edu
 Date: Thursday, November 12, 2009, 11:03 AM


 Hi All,
 I received an e-mail and an early morning call from my friend Helen Hedor
 this morning stating that she received a e-mail scam from me stating;
 FISHING FOR MONEY.  I did in NO WAY send this e-mail!  Please do not in
 anyway answer or send money to the bogus e-mail scam which was sent in my
 name..  THIS IS SCARY!
 I don't know what has happened, but I came home last night and my yahoo
 account sent me an e-mail which stated that my e-mail has been compromised
 for scam purposes, and if I didn't answer the questions below I would have
 my account frozen.  I immediately replied and answered the questions, but
 then I thought, what if this is a scam to extract personal information, so I
 copied the the questionnaire and sent it to the yahoo alert center.  I
 received a reply that they would check into it.  This morning I couldn't get
 into my e-mail account so I had to jump a bunch of hoops and reset my
 password.  I hope none of you have this happen to you.
 The Yahoo Account warning stated: Yahoo Mail has discovered series of
 illegal attempts on your Yahoo Account from bad Ip Location and will shut
 your account as it has been flagged as a spam account. Filling Correct
 Information Carefully and Sending to Yahoo Alert Center:
 I hope this was legitimate!  The information they wanted was my user name,
 password and birthday.
 Sincerely,Akemi Allison-Tacha BS, HT(ASCP)HTL
 E-Mail: akemiat3...@yahoo.com

 --- On Thu, 11/12/09, Helen Fedor hfe...@jhmi.edu wrote:

 From: Helen Fedor hfe...@jhmi.edu
 Subject: [Histonet] RE: Akemi - Urgent!
 To: Breeden, Sara sbree...@nmda.nmsu.edu, 
 histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
 Date: Thursday, November 12, 2009, 6:29 AM

 I also receive one of the scam emails.


 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu [mailto:
 histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Breeden, Sara
 Sent: Thursday, November 12, 2009 9:21 AM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Akemi - Urgent!

 Akemi Allison-Tacha - email me immediately, please.  I think you are
 being scammed and I don't want to email you directly.



 Sally Breeden, HT(ASCP)

 NM Dept. of Agriculture

 Veterinary Diagnostic Services

 PO Box 4700

 Albuquerque, NM  87106

 505-841-2576



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Re: [Histonet] Green Fluorescent Protein

[Adam .]

At the suggestion from someone on Histonet, I've been using Abcam's chicken
anti-GFP. It's worked great on mouse tissue fixed with paraformaldehyde, Zn
buffered formalin, or 10% neutral buffered formalin. Even though Abcam
suggests that you antigen retrieve, I've found it works just as well without
it.

As for viewing GFP without any antibody, you apparently can, at least in
bone. See
http://skeletalbiology.uchc.edu/30_ResearchProgram/304_gap/index.htm for a
really detailed discussion. I've tried before and it hasn't worked... I
think you need really high quality optics and filter cubes.

Adam

On Tue, Oct 27, 2009 at 8:50 AM, Paula Pierce 
cont...@excaliburpathology.com wrote:

 Hello,

 I routinely use Invitrogen's Rabbit anti-GFP on mouse and rat FFPE tissue.

 Paula




 
 From: Melanie Black melanie.bl...@uct.ac.za
 To: histonet@lists.utsouthwestern.edu
 Sent: Tue, October 27, 2009 7:48:48 AM
 Subject: [Histonet] Green Fluorescent Protein

 Hi

 I am looking to demonstrate Green fluorescent protein in Para formaldehyde
 fixed, processed rat tissue. Apart from using an antibody against GFP, can
 the native GFP be detected? Gayle Callis, I believe you may be able to help
 me with this method.

 Thanks
 Melanie

 Melanie Black
 082 469 3352

 Cardiovascular Research Unit
 3rd Floor; Chris Barnard Building
 Medical School;
 Observatory. 7925.
 University of Cape Town.
 South Africa.



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[Histonet] Isotype background

[Adam .]

Hi all,

I am trying some IHC, and I am having a peculiar problem. Like I expect, my
antibody of interest (anti-mouse goat polyclonal) stains nonspecifically at
high concentrations (10 ug / ml) but as I titer it down (3 ug / mL), it
seems to stain relatively specifically the cells I think it should stain.
However, at 3 ug / mL, my isotype goat IgG stains nearly everything.

Here is my protocol
1) Block in 3% H2O2 for 10'. Wash.
2) Block in 10% donkey serum for 1 hr. Wash.
3) Block in avidin 15', wash, biotin 15', wash (Vector Labs blocking kits)
4) Incubate with primary / isotype overnight at 4C in 2% donkey serum. Wash.
5) Incubate with secondary (biotinylated donkey anti-goat, cross adsorbed to
mouse) in 2% donkey serum for 1 hr at room temp. Wash.
6) Incubate with strepavidin HRP in TBS-T for 30'. Wash.
7) Incubate with DAB+ (Dako) for 5'.

For isotype, I am using Chrompure Goat IgG, whole molecule from Jackson.
Some people have suggested that I just do away with isotypes altogether and
use a no primary control instead. I think there is some merit to this idea,
but I still think my issue might be indicative of a larger technical problem
in my staining protocol.

Thanks,
Adam
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[Histonet] Going from immmunohistochemistry to immunofluorescence

[Adam .]

Hi all,

I have a few antibodies that I have successfully gotten to work using
immunohistochemistry (primary + biotinylated secondary + SA-HRP + DAB). I am
hoping to move from IHC to immunofluorescence. I tried the same staining
using either a fluorescently conjugated secondary or a biotinylated
secondary and an avidin conjugated fluorophore, but I don't see any staining
at all. I understand the peroxidase chemistry is a lot more sensitive than
fluorescence, so my question is, if I can get IHC to work, should I be able
to get IF to work? If so, how would I go about troubleshooting it? How
important is the flourophore (i.e. Alexa conjugates vs Texas Red vs
Rhodamine).

Yes, I know I could do dual immunohistochemistry, but I like fluorescence
since you can see each stain independently and then merge them
electronically. If anyone has a great suggestion on some chromagens that
work great together, I would welcome those suggestions as well.

Thanks,
Adam
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[Histonet] Staining with two primary antibodies from same host

[Adam .]

Hi all,

I'm looking into staining with two primary antibodies from the same host, in
this case goat. I've read a bit about this on Jackson Immunoresearch's
website, but I wanted to run by my idea to get an idea if this is at all
feasible.

I want to stain mouse tissue with antigen X and antigen Y. I have a two
polyclonal primaries: goat anti-mouse X and a goat anti-mouse Y. This is
what I was thinking

1) Block in donkey serum for 1 hr at room temp.
2) Incubate with goat anti-mouse X overnight at 4C. Wash.
3) Incubate with Dylight 488 donkey anti-goat for 30 mins at room temp.
Wash.
4) Reblock in donkey serum for 1 hr at room temp.
5) Incubate with 10 - 20 ug / mL unconjugated donkey anti-goat Fab for 1 hr
at room temp -- cheapest I could find was at
Rocklandhttp://www.rockland-inc.com/ccp8033-fab-fragment-of-affinity-purified-anti-goat-igg-28-805-7102-805-7102.htm.
Wash.
6) Incubate with goat anti-mouse Y overnight at 4C. Wash.
7) Incubate with biotin donkey anti-goat for 30 mins at room temp. Wash.
8) Incubate with avidin AMCA for 30 mins at room temp

Would this work? Is there an easier or better way? What are the pitalls or
tips you could offer?

Hope you all aren't reading this during your long weekend,
Adam
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Re: [Histonet] Re: anti-GFP antibody

[Adam .]

Hi all,

Just to update you, I ordered Abcam's ab13970 chicken anti-GFP and tested it
out in paraffin embedded sections with HIER for 10 minutes in Dako target
retrieval system. I came in with a Dylight 488 secondary from Jackson
Immunoresearch at 1 ug / mL. It worked beautifully at 1:500, 1:1000, and
1:2000. I should've titered it even further down.

Thanks for all your help,
Adam

On Thu, Aug 20, 2009 at 1:38 PM, Hobbs, Carl carl.ho...@kcl.ac.uk wrote:


 I am sure someone has a cunning combination!
  I use Abcam's ab13970 chicken anti  GFP in Pwax sections after HIER.
 For me, it is very good
 Image at Abcam or here : http://www.immunoportal.com/index.php
 Good luck!
 carl



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Re: [Histonet] uneven alternating sections on cryostat

[Adam .]

I was having this problem too, but I think I finally figured it out. There
is a screw on our cryostat that attaches the chuck to the rest of the
machine. This screws fits into a small hole in the chuck. Sometimes the
screw isn't well set in the hole or is well set but for some reason comes
loose and causes the chuck to wobble. For some reason, this causes your
problem. Now I make sure that everything is tightly secured, and I rarely
have this problem.

Good luck,
Adam

On Thu, Aug 27, 2009 at 4:47 PM, Nathan Cramer natec...@gmail.com wrote:


   Thanks, Robyn... I'll make sure the holder is clamped down. (sometimes
   its the little things...)
   Best
   Nate
R J VAZQUEZ wrote:

 Nathan,
 It sounds like the blade holder is not secure enough or even in the
 tightness on each side.
 Hope this helps.
 Robyn

  Date: Thu, 27 Aug 2009 16:44:11 -0400
   From: [1]natec...@gmail.com
  To: [2]histo...@lists.utsouthwestern.edu
   Subject: [Histonet] uneven alternating sections on cryostat
 
  When cutting PFA fixed, cryoprotected tissue on our cryostat, I
   frequently  find  that  every other section gets cut improperly.
 I'll get
   one nicely cut section and then on the next pass I only get half
 of a
   section. This cycle simply repeats over and over and I lose many
 slices.
  It has been a while since our cryostat has been serviced, so I'm
   wondering  if  this  an  operator  error or a machine problem. I
 thought
   maybe  the  tissue  temperature hadn't settled properly, but the
 uneven
   cutting  still  happens  even  when  I let the tissue sit for 30
 minutes in
  the chuck holder. (cutting mouse spinal cord at -20C)
 
   On another note, if anyone has any tips for improving white/gray
 matter
   contrast  in frozen spinal cord sections stained with luxol fast
 blue,
   I'd  be very appreciative. I do defat the slices with chloroform
 and
   differentiate  with  lithium carbonate but everything is usually
 either
   very  dark  or  very  light. I am learning as I go (checking the
 archives
  here often) so any help would be great.
 
  Thanks!
 
  Nathan Cramer
  Neurobiology and Behavior
  Cornell University
  Ithaca, NY 14853
 
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 References

   1. mailto:natec...@gmail.com
   2. mailto:histonet@lists.utsouthwestern.edu
   3. mailto:Histonet@lists.utsouthwestern.edu
   4. http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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[Histonet] Cleaning oil off objectives

[Adam .]

Hi all,

You guys were so helpful on my last question, I'll ask another. We have a
microscope shared by the floor with several objectives, and it's pretty
common for the non-immersion objectives to get contaminated with oil. I
asked the guy who is responsible for the scope about this. He said that they
call someone from some company who carefully cleans the objectives with
acetone and a Q-tip, which if done right works wonders but if done wrong it
can damage the lenses. But he mentioned that the lenses are usually
re-contaminated within a few weeks since so many people use the scope, so
it's sort of a pointless endeavor. This system seems pretty silly to me... I
feel like there must be an easier and cheaper way to clean the lenses
without damaging them; I certainly don't want to be responsible for damaging
a microscope that costs more than my yearly salary. What do you recommend?

Thanks,
Adam
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[Histonet] anti-GFP antibody

[Adam .]

Hi all,

I am looking for a good anti-GFP antibody. I plan on using it for dual
immunofluorescence on mouse bone in paraformaldehyde fixed, paraffin
embedded sections, most likely with some antigen retrieval.

Here's the main problem: I want to use it with a goat-anti-mouse primary and
in a separate assay with a rabbit-anti-mouse antibody, both of which are
staining mouse bone. In order to save money and optimization time, I would
prefer if it were raised in a species other than goat and rabbit so I could
use it for both assays. I also want to avoid mouse antibodies to avoid the
mouse-on-mouse issue. Alternatively, I could buy two antibodies (rabbit and
goat) and use them in the respective assays. Or is there another way that's
non-obvious?

Thanks,
Adam
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[Histonet] 4F:1G

[Adam]

I'm new to the field of histology and want to use 4F:1G to fix fish
ovaries for later histological examination under light microscopy.
Following fixation, I plan to rinse the ovary samples in an ethanol
series (50-95%).  First, what is the minimum time needed to fix 10 g
of tissue?  Second, I would like to store the fixed samples for a few
months prior to embedding.  Is it best to maintain them in 4F:1G or to
keep them in ethanol after the rinses?

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[Histonet] Cryoprotection Issues with Unfixed Mouse Hearts

[Adam Bazama]

Does anyone have any suggestions or protocols for cryoprotecting unfixed
mouse heart tissue? Some of the ones I have been cryosectioning lately have
been damaged, I think because of improper cryoprotection.

 

Thanks!

 

Adam

baza0...@umn.edu

 

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[Histonet] UC Berkeley students in need of help

[Adam Anthony]

Fellow Histoneters,

Can you spare less 2 minutes of your time to help out a group of 4 MBA students 
at UC Berkeley?  We are conducting market research, by way of a simple online 
survey, for a class project.  Our focus is on ways in which labs can cut 
significant costs on reagents and materials.  There are 11 short questions- 
please help if you have time.

Link to online survey:
http://www.surveymonkey.com/s.aspx?sm=X3ssptgNq4oh7o0vwy1gCg_3d_3d

Please contact me with any questions.

Thanks,
Adam Anthony
adam_anth...@mba.berkeley.edumailto:adam_anth...@mba.berkeley.edu
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[Histonet] HIF-1alpha or Ref-1 immunohistochemistry on mouse FFPE tissues

[Adam Bazama]

Does anyone have any suggestions or a working immunofluorescence protocol
for staining mouse FFPE tissues with HIF-1alpha rabbit polyclonal or Ref-1
rabbit polyclonal? Both from Santa Cruz.

 

Thank you, Adam

 

Adam Bazama

baza0...@umn.edu

Lillehei Heart Institute 

Histology Microscopy Research Core

University of Minnesota
4-266 Nils Hasselmo Hall
Minneapolis, MN 55455
612-625-6779

 

 

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[Histonet] Cutting fresh-frozen brains from 1 week old rat pups

[Adam Galle]

Hi all,
Currently I am working with brains from 7 day old rat pups, that undergo an 
hypoxic-ischemic injury (Levine/Vannucci technique). These brains are 
unfixed, frozen in isopentane and cut at 20um on a croystat. These brains 
are not cutting very well compared to an adult brain (potenially due to 
unfinished myelination?), they are very 'crumbly' for want of a better term 
and always have cracks or generally poor preservation of morphology. I have 
tried all the standard tricks of different temperatures, section thickness 
and knife angle to no avail. I am going to perfuse fix my next cohort of 
animals with PFA and then a 30% sucrose step to see if that helps, but I was 
hoping that someone out there would have some tips on cutting these immature 
brains.


Thanks,
Adam.


Adam Galle,

Neuropharmacology and Brain Injury Lab
Department of Pharmacology
School of Medical Sciences
UNSW Sydney



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[Histonet] sirius red connective tissue stain[Scanned]

[Adam Bazama]

Hi Dave,

 

I noticed that you posted a request in march of 2004 on histonet for a
Sirius red fast green staining protocol. If you do have a protocol, would
you mind sending it to me. I have had a hard time finding one to compare to
mine and my lack luster results.

 

Thank you,

 

 

Adam Bazama, B.S.

Junior Scientist

Lillehei Heart Institute Histology and Microscopy Core Facility

University of Minnesota Medical School, Division of Cardiology
4-266 Nils Hasselmo Hall
312 Church Street SE
Minneapolis, MN  55455
Lab Desk: 612-625-6779
Cell: 952-334-0607

 

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