[Histonet] unsubscribe

[Pauletta A. Robbins via Histonet]

Please unsubscribe my email address
Thank you!
Pauletta A. Robbins
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Re: [Histonet] Suggestions for making glutaraldehyde penetrate in between little setae of insects for microscopy

[Jay Lundgren via Histonet]

A little alcohol will lower the surface tension of aqueous solutions.  Then
there's always DMSO.


   Sincerely,

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

On Fri, Feb 26, 2016 at 8:15 AM, Jorge A. Santiago-Blay via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello Histonetetrs:
>
> I am trying to fix insect wings for TEM. Less than ideal fixation appears
> to be caused by abundant setae on the surface of the wings. Those setae,
> trap air, thus making penetration of the fixative more difficult difficult.
> Interestingly, also the wings tend to curl upon themselves.
>
> Options re. fixation:
> 1. Just submerging the insects mechanically does not seem to solve the
> problem.
>
> 2. We are contemplating submerging and evacuating (intermittently) a few
> times for 2 minutes (vacuuming)
>
> 3. Are there chemicals that can break the bubbles formed in between the
> setae?
>
> 4. Any other comments or suggestions?
>
> Options to minimize (ideally eliminate) curling of the wings?
>
> If you have any constructive suggestions, please kindly email it to me
> directly.
>
> blayjo...@gmail.com
>
> Of course, we intend to begin with new freshly collected insects.
>
> Gratefully,
>
> Jorge
>
> Jorge A. Santiago-Blay, PhD
> blaypublishers.com
>
> 1. Positive experiences for authors of papers published in *LEB*
> http://blaypublishers.com/testimonials/
>
> 2. Free examples of papers published in *LEB*:
> http://blaypublishers.com/category/previous-issues/.
>
> 3. *Guidelines for Authors* and page charges of *LEB*:
> http://blaypublishers.com/archives/ *.*
>
> 4. Want to subscribe to *LEB*? http://blaypublishers.com/subscriptions/
>
>
> http://blayjorge.wordpress.com/
> http://paleobiology.si.edu/staff/individuals/santiagoblay.cfm
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Re: [Histonet] Suggestions for making glutaraldehyde penetrate in between little setae of insects for microscopy

[Simmons, Christopher via Histonet]

Double PPE if you use DMSO


Sent from my iPhone

> On Feb 26, 2016, at 2:41 PM, Jay Lundgren via Histonet 
>  wrote:
> 
> A little alcohol will lower the surface tension of aqueous solutions.  Then
> there's always DMSO.
> 
> 
>   Sincerely,
> 
>Jay A. Lundgren, M.S., HTL (ASCP)
> 
> On Fri, Feb 26, 2016 at 8:15 AM, Jorge A. Santiago-Blay via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
> 
>> Hello Histonetetrs:
>> 
>> I am trying to fix insect wings for TEM. Less than ideal fixation appears
>> to be caused by abundant setae on the surface of the wings. Those setae,
>> trap air, thus making penetration of the fixative more difficult difficult.
>> Interestingly, also the wings tend to curl upon themselves.
>> 
>> Options re. fixation:
>> 1. Just submerging the insects mechanically does not seem to solve the
>> problem.
>> 
>> 2. We are contemplating submerging and evacuating (intermittently) a few
>> times for 2 minutes (vacuuming)
>> 
>> 3. Are there chemicals that can break the bubbles formed in between the
>> setae?
>> 
>> 4. Any other comments or suggestions?
>> 
>> Options to minimize (ideally eliminate) curling of the wings?
>> 
>> If you have any constructive suggestions, please kindly email it to me
>> directly.
>> 
>> blayjo...@gmail.com
>> 
>> Of course, we intend to begin with new freshly collected insects.
>> 
>> Gratefully,
>> 
>> Jorge
>> 
>> Jorge A. Santiago-Blay, PhD
>> blaypublishers.com
>> 
>> 1. Positive experiences for authors of papers published in *LEB*
>> http://blaypublishers.com/testimonials/
>> 
>> 2. Free examples of papers published in *LEB*:
>> http://blaypublishers.com/category/previous-issues/.
>> 
>> 3. *Guidelines for Authors* and page charges of *LEB*:
>> http://blaypublishers.com/archives/ *.*
>> 
>> 4. Want to subscribe to *LEB*? http://blaypublishers.com/subscriptions/
>> 
>> 
>> http://blayjorge.wordpress.com/
>> http://paleobiology.si.edu/staff/individuals/santiagoblay.cfm
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Re: [Histonet] Image analysis software

[Elizabeth Chlipala via Histonet]

Judi

Through the years we have used a wide variety of software applications, we have 
been performing image analysis since 2006, we started whole slide scanning in 
2007.  We have used various modules from Zeiss, Image Pro, Aperio's Toolbox, 
Definiens and free shareware such at Image J.  There are also new vendors on 
the market such as Indica Labs and Visopharm.  I think you need to make an 
educated decision based upon what your needs are and how complex your analysis 
is and the price of the product, some of the commercially available software 
packages can be expensive.  I saw the earlier post that discussed Image J and 
that might be a good option to start with if you have the time and ability to 
learn how to work with the software, you would be surprised to learn that Image 
J can do quite a bit.  The only thing that it may not be capable of is pattern 
recognition, others that use the software can comment on that aspect.  I do 
know that it is easier to trace in Image J than it is in some of
  the viewer software that are linked to a particular scanner. 

The other software solutions may be a bit easier to understand and work with 
initially but there will always be a learning curve associated with any 
software package that you choose to use.  Most software packages will support 
different file types, such as tiff or modified tiff, you will need to make sure 
the image format your scanner generates is compatible with the software you 
choose.   My suggestion would be to look at all of them and determine what is 
best for your use cases.  I would also be a bit forward thinking, you might 
think that you only need the software to do certain things but as you use the 
technology more you will see that you will want to use if for other use cases.  

The other thing I want to comment on is that the success of your image analysis 
is not just related to the software that you choose to use  but also the 
quality, consistency and reproducibility of your histology preparations.  

If you want more information on the topic I would suggest going to the DPA - 
Digital Pathology Association website, it has a list of most of the vendors in 
the space and if you become of member, its $50.00 for technologists, you will 
have access to all of the past presentations from the Pathology Visions 
conferences and webinars.  Here is the link to the website.  The other option 
is to attend the Pathology Visions conference, it a conference geared towards 
digital pathology.  If you are a NSH member there is a scholarship available to 
attend the conference, you could apply for that.  The links to the NSH 
scholarship and the Pathology Visions conference are below. The list of vendors 
is under the resources tab.  

https://digitalpathologyassociation.org/

https://digitalpathologyassociation.org/pathology-visions-2016

http://nsh.org/content/digital-pathology-association-scholarship-pathology-vision

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: Judi Ford via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, February 25, 2016 5:36 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Image analysis software

Hi everyone,
We are in the middle of looking at image analysis software and I was wondering 
what software programs people are using. Why did you pick the one you are 
using? Are you using it for brightfield whole slide analysis or for fluorescent 
slide analysis?  What file types can be used with your software?

I would love to hear your experiences.

Thanks,
Judi
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[Histonet] h. pylori

[Dooley, Elaine via Histonet]

Hi histonetters,

We filter the H.pylori antibody with the filters that come with the Ventana 
Prep kits.

Elaine Dooley
Shands Teaching Hospital
352-265-0111 ext 72117
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[Histonet] H. pylori by IHC

[Joanne Clark via Histonet]

Recently someone placed a query regarding the precipitate with Cell Marques H. 
pylori marker.  The response was to filter it before use to clean it up.  May I 
ask how it is being filtered?  We have the same problem and it makes the slide 
difficult to interpret.


Joanne Clark, HT BAAS
Director of Histology
Pathology Consultants of New Mexico



Disclaimer: This electronic message may contain information that is proprietary,
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[Histonet] Friday Fun (non-work related blog post, easy to ignore)

[Lester Raff MD via Histonet]

Anyone moving to Chicago area?

http://www.chicagonow.com/downsize-maybe/2016/02/to-the-family-who-will-buy-our-house-whoever-you-are/


Lester J. Raff, MD MBA
UroPartners
Medical Director Of Laboratory
2225 Enterprise Dr. Suite 2511
Westchester, Il 60154
Tel: 708-486-0076
Fax: 708-492-0203

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Re: [Histonet] CAP

[Elizabeth Chlipala via Histonet]

Bernice

We are not a clinical lab, we are a GLP compliant lab and we have a procedure 
that addresses this and everything else about reagent preparation.  I will put 
the procedure and forms that we use on the NSH BLOCK for anyone who is 
interested.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-Original Message-
From: Bernice Frederick via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, February 26, 2016 6:57 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] CAP

ANP21382. What kind of policy or SOP do you all have for this question? T asks 
how reagents are given an expiration. This includes but is not restricted to 
reagents where manufacturer does not specify a date. We date made up reagents 
as a 6 month expiration unless we know it doesn't last that long. Came up 
during interim self inspection.
Thanks,
Bernice

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723
b-freder...@northwestern.edu

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[Histonet] glycine method to remove aldehyde induced autofluorescence

[Gayle Callis via Histonet]

Dear All, 

 

Some person kindly mentioned my name as a source for the glycine method to
remove aldehyde induced autofluorescence.  We liked the simplicity of this
method, plus gentle to tissue sections.  

 

This was the original information but we modified it.   I have seen
concentrations of glycine range from 100 mM to 700 mM . 

 

Original method:  
 
1. Rehydrated tissue sections:  A Tris-glycine mixture (adjust 0.1M glycine
to pH 7.2-7.4 with 1M Tris base will saturate free aldehyde groups. (15-30
minutes at room temp in Tris-glycine for FFPE sections. Wash well in PBS.
If the tissue is fragile though, only use the Tris-glycine method.
 
2..  For tissues coming out of formalin, soak the tissues for 30 min to 1
hour and rinse well. 

 

 

Callis Modified Method: 

 

We did not use TRIS-glycine, preferring the same buffer used in IF staining.
Make fresh for a day's use.   500 mM glycine in pH 7.2 - 7.4  Dulbeccos PBS
(Sigma).  We increased concentration to 500 mM glycine for 15 - 30 minutes
at RT after we found 100 mM reduced autofluorescence while the higher
concentration did a better more complete removal.I don't think it makes
much difference if you use TBS or DPBS so you can use whatever your lab
prefers for IHC/IF staining should work equally well. 

  

We found  two changes of glycine solution worked well since you are
refreshing the solution to sop up those free aldhydes.  Do 15 minutes
incubation for each change, don't  rinse the sections between changes, just
tip, drain slides, blotted edges of sections, add solution on sections with
slides laying flat on a manual stainer.   Some people might prefer glycine
solution in a coplin jar if they are going to an automated staining system.


 

If you fear drying, one method was 700mM glycine, 0.15% BSA (use pure IgG
and protease free), and 0.1% sodium azide in PBS with 15 to 30 min RT
incubation.   Sodium azide can be left out since it is there to prevent
bacterial growth, and deemed unnecessary since our glycine solution was made
fresh before a one day/one time use.  Glycine is cheap and goes into
solution readily.  

 

FYI, lysine has also been used to get rid of free aldehydes (Elias J.
Immunohistopathology book) 

 

Good luck

 

Gayle Callis

HTL/HT/MT(ASCP)  

Bozeman MT   

 

 

 

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Re: [Histonet] auto-fluorescence

[Gayle Callis via Histonet]

Dear Joost, 

Yes and to read up on this, go to this website for an excellent, well
referenced free pdf i.e. Autofluorescence, causes and cures.
https://www.google.com/?gws_rd=ssl#q=autofluorescence+causes+and+cures+wrigh
t+cell+imaging+facility   They also posted a pdf on mounting medias for
fluorescence microscopy work.  

The pdf will tell you what tissue and cellular components autofluoresce.
The authors also provided various methods to reduce or remove
autofluorescence of these components but also how to remove aldehyde induced
autofluorescence if you are fixing tissues with NBF or PFA.   

Remember there is no autofluroescence in the Near Infra Red region so one
can use an NIR fluorophore, i.e. Alexa 750 (red) or another fluorophore for
the NIR region,  and even use tissue autofluorescence as a "counterstain
fluorescence" However, you cannot see these with the human eye but these
photograph beautifully.  OR if you have a spectral imaging or confocal
capabilities,  you can rule out autofluorescence without having to treat the
tissue section.

If you have difficulty access this pdf, I will send via private email.  

Gayle Callis HTL/HT/MT(ASCP)  
Bozeman MT USA



-Original Message-
From: Bruijntjes, J.P. (Joost) via Histonet
[mailto:histonet@lists.utsouthwestern.edu] 
Sent: Friday, February 26, 2016 3:00 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] auto-fluorescence

Hi histonetters

I hope one of you can help me. I don't have any experience with
fluorescence.

We are searching for some components and we will use  an alexa488 conjugated
secondary. Will there be an auto-fluorescence on different organs
(brain/spleen/liver)?

Greetings
Joost



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[Histonet] HISTOPALOOZA III Georgia Society for Histology

[Zimmerman, Billie via Histonet]

Histopalooza! III welcomes Ely Klar, MS,HTL(ASCP) back as a presenter.  Ely is 
an instructor at Columbus State University in Columbus, Georgia, home of the 
Cougars.  (Cougars being the school mascot, not where all single women over 40 
live.)  Her workshop on microscopic anatomy is always well attended.  It's a 
great workshop for someone sitting for the HT/HTL exams or for anyone that 
needs a refresher. Ely has a warm personality and people truly enjoy her 
presentation.

Warm regards,
Billie Zimmerman  (too old to be a cougar)
GSH secretary

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[Histonet] Suggestions for alternative fluorescent nuclei stains (that are not DAPI) ?

[Michael Bruno via Histonet]

Hello all,

I perform immunohistochemistry (IHC) and immunofluorescence (IF) on 
fixed free-floating rat brain sections (~50um thickness).  Currently I 
use Prolong Gold with DAPI as a mounting media as well as a stain for 
the nuclei.


I am seeking an alternative (fluorescent) nuclei stain for free floating 
rat brain sections.  The issue I am having is that one of my fluorescent 
markers (FluoroGold) has native fluorescence in the UV channel.  Since 
it is the same channel that DAPI is in, this will not do.  I do not have 
an antibody or fluorescent marker in the far-red channel so I would 
really like to find one in that area.


I have looked at To-Pro-3 and NucRed Dead 647 as alternatives because 
their fluorescence is in the far-red channel. However I am unclear if 
these stains can be used on brain sections and not just in vitro cells.


To summarize, I am looking to find an alternative nuclei stain to DAPI, 
and one that is preferably in the far-red channel.  If anyone here has a 
protocol for a nuclei stain that they would not mind sharing, I would be 
very appreciative as well.


Thank you so much for your help,

Mike Bruno
State University of New York at Buffalo

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[Histonet] Suggestions for making glutaraldehyde penetrate in between little setae of insects for microscopy

[Jorge A. Santiago-Blay via Histonet]

Hello Histonetetrs:

I am trying to fix insect wings for TEM. Less than ideal fixation appears
to be caused by abundant setae on the surface of the wings. Those setae,
trap air, thus making penetration of the fixative more difficult difficult.
Interestingly, also the wings tend to curl upon themselves.

Options re. fixation:
1. Just submerging the insects mechanically does not seem to solve the
problem.

2. We are contemplating submerging and evacuating (intermittently) a few
times for 2 minutes (vacuuming)

3. Are there chemicals that can break the bubbles formed in between the
setae?

4. Any other comments or suggestions?

Options to minimize (ideally eliminate) curling of the wings?

If you have any constructive suggestions, please kindly email it to me
directly.

blayjo...@gmail.com

Of course, we intend to begin with new freshly collected insects.

Gratefully,

Jorge

Jorge A. Santiago-Blay, PhD
blaypublishers.com

1. Positive experiences for authors of papers published in *LEB*
http://blaypublishers.com/testimonials/

2. Free examples of papers published in *LEB*:
http://blaypublishers.com/category/previous-issues/.

3. *Guidelines for Authors* and page charges of *LEB*:
http://blaypublishers.com/archives/ *.*

4. Want to subscribe to *LEB*? http://blaypublishers.com/subscriptions/


http://blayjorge.wordpress.com/
http://paleobiology.si.edu/staff/individuals/santiagoblay.cfm
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[Histonet] CAP

[Bernice Frederick via Histonet]

ANP21382. What kind of policy or SOP do you all have for this question? T asks 
how reagents are given an expiration. This includes but is not restricted to 
reagents where manufacturer does not specify a date. We date made up reagents 
as a 6 month expiration unless we know it doesn't last that long. Came up 
during interim self inspection.
Thanks,
Bernice

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723
b-freder...@northwestern.edu

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[Histonet] Suggestions for staining bacteria in FFPE decaled growth plate

[Anna Huntley Coffey via Histonet]

Happy Friday, Histonetters!

I have a puzzle that I could really use your expert knowledge on this
morning. One of our pathologists has a decalcified FFPE mouse bone embedded
longitudinally and has found some bacteria located in the growth plate. The
bacteria has been confirmed with fluorescence, but she really wants to be
able to show the morphology of the tissue with the bacteria. So far, H,
gram, Brown & Hopps stain the growth plate so dark that you can't make out
the bacteria. My suggestions were to try a thinner section and adjust the
timing of the H (haven't had a chance to do that yet), but I wanted to
throw the question out to you all to see if anyone has tried other stains
or techniques for this type of issue.

I'd really appreciate the help!

Thanks,
Anna
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[Histonet] Direct Hire for Histotech Job in Houston, TX

[Melissa Owens via Histonet]

Happy Friday Everyone!

I have a position in Houston, TX for Full Time/Permanent. The shift is Evening 
Shift. Please contact me for job description. Have a great day!

Melissa Owens
President, Laboratory Staffing
Allied Search Partners

T: 888.388.7571 ext. 102

F: 888.388.7572


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Re: [Histonet] Image analysis software

[Julio Benavides via Histonet]

Hi Jodi,

I was using imageAnalysis from Olympus some years ago, bus since I  
discovered ImageJ there is no way I will go back to commercial stuff.  
Pros of ImageJ, apart from the obvious one that it is free, is that a  
lot of people is using it, so plenty of tutorials, tricks, how-to in  
internet. Also, there are plenty of add-on to this software so there  
will be always one application for your needs. You don't need a big  
computer to work with it. Only downside, if you can consider it, is  
that the looks of the software are not as fancy as the commercial ones.


I use it for image analysis in IHC and IHF.

Hope this helps. i also would be happy to know what other people  
thinks regarding this issue.


Cheers

Julio



Judi Ford via Histonet  escribió:


Hi everyone,
We are in the middle of looking at image analysis software and I was  
wondering what software programs people are using. Why did you pick  
the one you are using? Are you using it for brightfield whole slide  
analysis or for fluorescent slide analysis?  What file types can be  
used with your software?


I would love to hear your experiences.

Thanks,
Judi
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Re: [Histonet] Human nuclear antibody MAB1281

[Hobbs, Carl via Histonet]

I had/have the same problem with that ab on FFPWS +/- AR
Also negative, under same conditions:
Serotec's 6970-1257, Abcam's ab5675

I use 2 abs on hu teratomas in mouse host:

Takara clontech STEM121 antibody
Abcam anti MTCO2 ab110258

Images to be seen on Histonet Images site




  
 
Carl Hobbs FIBMS 
Histology and Imaging Manager 
Wolfson CARD 
Guys Campus, London Bridge  
Kings College London 
London 
SE1 1UL 
  
020 7848 6813
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