[Histonet] C4d

[Algeo, Lacie A]

Hi Everyone,
Does anyone know of a FITC C4d that is ASR or IVD?  All I have been able
to find is RUO.  Also, is anyone using something different to show
rejection in transplant kidneys?
Thank you,
Lacie

Lacie Algeo, HTL, MP(ASCP)
Histology Supervisor
Providence Sacred Heart Medical Center Laboratory
101 W 8th Avenue
Spokane, WA 99220
509-474-4418
FAX 509-474-2052
lacie.al...@providence.org

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[Histonet] used microscope vendors

[Caroline Bass]

Can anyone recommend a good source for either used microscopes or an 
inexpensive brand? I'm looking for a basic inverted microscope for very basic 
cell counts, and quick looks for cell viability and health. This is very much a 
quick in and out scope for students to kick around and abuse. I'm purchasing a 
fluorescent microscope for more extensive needs. I've looked on ebay and a 
couple other places, but the market is inundated with scopes that it makes it 
difficult to figure out. 

Ideally, I'd love a recommendation for a basic used scope and maybe some names 
of reputable vendors of refurbished units.

Thanks,

Caroline
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RE: [Histonet] Uni-trieve, antigen retrieval for immunofluorescence

[Andrea Marion]

Hi Sarah,

Thanks for the info! Have you used Unitrieve for immunofluorescence? I am
wondering whether I will see a reduction in the autofluorescence caused by
heating? I will request a sample to try and report back to all.

Andrea

Andrea Marion
Graduate Student
University of Illinois at Chicago

On Tue, September 20, 2011 12:49 pm, sdys...@mirnarx.com wrote:
> I actually attended a class they put on a few months ago where they used
> Unitrieve.  It works really well.  It doesn't seem to produce any kind
> of background and yes, it is used at much lower temps.  However the
> retrieval process takes a little longer, but...you can put it in an even
> lower temp. water bath overnight, come in to work in the morning, and
> your slides are reading to go.
> Contact Zara over there and she might be able to hook you up with a
> sample.
> Good Luck!!
>
> Sarah Goebel-Dysart, BA, HT(ASCP)
> Histotechnologist
> Mirna Therapeutics
> 2150 Woodward Street
> Suite 100
> Austin, Texas  78744
> (512)901-0900 ext. 6912
>
>
> -Original Message-
> From: histonet-boun...@lists.utsouthwestern.edu
> [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea
> Marion
> Sent: Tuesday, September 20, 2011 11:59 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Uni-trieve, antigen retrieval for immunofluorescence
>
> Hello all,
>
> Has anyone used the reagent 'Uni-trieve' from Innovex? It is purported
> to
> be a universal antigen retrieval solution that can be used at lower
> temperatures (65-70 C for cytoplasmic antigens, and 75-80 C for nuclear
> antigens):
>
> http://innvx.com/unitrievepage.html
>
> The company claims that the reagent is a universal retrieval solution
> for
> all antibodies and tissues (which is silly of course - how could they
> know?). Does anyone have any experience with the product?
>
> I am interested because I see that increased heat during antigen
> retrieval
> causes greater tissue autofluorescence during immunofluorescence
> stainings. My current protocol is to use 20 minutes at 90-95 C on a hot
> plate using sodium citrate buffer. Does anyone else either Uni-trieve or
> a
> different reagent/protocol for immunofluorescence stainings?
>
> Thanks,
>
> Andrea
>
> Andrea Marion
> Graduate Student
> University of Illinois at Chicago
>
>
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>
>



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[Histonet] Tissue handling at embedding

[Tim Higgins]

I have worked at labs that reuse molds and ones that clean between uses.
Guess it all depends on what your definition of a "clean" mold is.  If you
have the time and space, not reusing the molds is best practice but not
always practical.

Thanks,

Tim


Message: 10
Date: Tue, 20 Sep 2011 09:44:26 -0700
From: "Morken, Timothy" 
Subject: [Histonet] Tissue handling at embedding
To: "histonet@lists.utsouthwestern.edu"

Message-ID:
<8d7c2d242dbd45498006b21122072bf85e2ae...@mcinfrwem003.ucsfmedicalcenter.org
>

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

What procedures do you have in place to prevent tissue loss at embedding?
What do you do if tissue appears to have been lost?

And do you clean embedding molds before each reuse, or after one-days use
(which may be many re-uses)?


Thanks for all info!


Tim Morken
Supervisor, Histology, IPOX
UC San Francisco Medical Center
Box 1656
1600 Divisidero St, B217
San Francisco, CA 94115
USA

415.514.6042 (office)
415.885.7409 Fax
tim.mor...@ucsfmedctr.org




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[Rene J Buesa]

Under separate cover I am sending something I wrote about this subject.
René J.

--- On Tue, 9/20/11, Jennifer MacDonald  wrote:


From: Jennifer MacDonald 
Subject: [Histonet] cost of an H&E
To: histonet@lists.utsouthwestern.edu
Date: Tuesday, September 20, 2011, 2:23 PM


Has anyone calculated the approximate cost of an H&E, materials only, no 
labor?  Including the cost of the cassette, paraffin, slide, hematoxylin, 
eosin and staining reagents?
Thanks,
Jennifer MacDonald
Mt. San Antonio College
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Re: [Histonet] Tissue handling at embedding

[Rene J Buesa]

This is how we do it:
1- the person grossing will dictate how many pieces should go into each cassette
2- the person cassetting will check that information against the vial with the 
pieces and write down in the cassettes summary form the number of pieces
3- the person embedding checks the pieces in the cassette after processing 
against the cassettes log.
If any piece is missing between any two steps, it will be looked for. Sometimes 
is small and is in the vial before cassetting, or small and in the retort.
Any irregularity is documented in the QA log.
René J.

--- On Tue, 9/20/11, Morken, Timothy  wrote:


From: Morken, Timothy 
Subject: [Histonet] Tissue handling at embedding
To: "histonet@lists.utsouthwestern.edu" 
Date: Tuesday, September 20, 2011, 12:44 PM


What procedures do you have in place to prevent tissue loss at embedding? What 
do you do if tissue appears to have been lost?

And do you clean embedding molds before each reuse, or after one-days use 
(which may be many re-uses)?


Thanks for all info!


Tim Morken
Supervisor, Histology, IPOX
UC San Francisco Medical Center
Box 1656
1600 Divisidero St, B217
San Francisco, CA 94115
USA

415.514.6042 (office)
415.885.7409 Fax
tim.mor...@ucsfmedctr.org


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[Histonet] Effect of Freezing on Unfixed GFP

[Andrew Coleman]

We have performed lentiviral injections with a turboGreenFluorescentProtein
(tGFP) into the brains of rats for an RNAi experiment. We did not fix the
tissue but froze in isopentane on dry ice and stored the tissue at -80 deg
Celsius.

We want to use some sections for a reference of the injection site (GFP
fluorescence) relative to the brain morphology (Neurotracer staining) in
order to selecitvely laser microdissect (LMD) infected cells from other
sections.

Our initial studies showed that fixation of sections on slides in 4%
paraformaledhyde (PFA) at 4 deg C for 5 minutes provided tGFP flourescence
comparable to that found in perfused tissue, but lately we see very weak or
no fluorescence with this procedure, even when increasing the time.

The only difference I can think of that we saw best results soon after the
brains were removed (ie 1 to 2 weeks) and now we are having trouble
visualizing the tGFP ~2 months of the brains being stored at -80 degrees
celsius.

Does anyone have experience with or an explanation of fluorescence
decreasing with a frozen, unfixed GFP over time?

In the future we plan on doing these experiments including a perfusion with
a low conc of PFA (to preserve RNA integrity) but for now we are trying to
figure out how to identify the GFP-positive cells in the animals we have
already performed surgery on. Another option would anti-tGFP IHC, but we
would prefer at this point to use on slide-fixed sections as a references
for our LMD.

Thanks!  -Andrew
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histonet@lists.utsouthwestern.edu

[Jennifer MacDonald]

Has anyone calculated the approximate cost of an H&E, materials only, no 
labor?  Including the cost of the cassette, paraffin, slide, hematoxylin, 
eosin and staining reagents?
Thanks,
Jennifer MacDonald
Mt. San Antonio College
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histonet@lists.utsouthwestern.edu

[Burton, Lynn]

We calculated that to be right around $4.05 per slide. That does not include 
labor.

Lynn Burton
Lab Assoc I
Animal Disease Lab
Galesburg, Il
309-344-2451

From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jennifer MacDonald 
[jmacdon...@mtsac.edu]
Sent: Tuesday, September 20, 2011 1:23 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] cost of an H&E

Has anyone calculated the approximate cost of an H&E, materials only, no
labor?  Including the cost of the cassette, paraffin, slide, hematoxylin,
eosin and staining reagents?
Thanks,
Jennifer MacDonald
Mt. San Antonio College
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RE: [Histonet] Uni-trieve, antigen retrieval for immunofluorescence

[sdysart]

I actually attended a class they put on a few months ago where they used
Unitrieve.  It works really well.  It doesn't seem to produce any kind
of background and yes, it is used at much lower temps.  However the
retrieval process takes a little longer, but...you can put it in an even
lower temp. water bath overnight, come in to work in the morning, and
your slides are reading to go.
Contact Zara over there and she might be able to hook you up with a
sample.
Good Luck!!

Sarah Goebel-Dysart, BA, HT(ASCP)
Histotechnologist
Mirna Therapeutics
2150 Woodward Street
Suite 100
Austin, Texas  78744
(512)901-0900 ext. 6912


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea
Marion
Sent: Tuesday, September 20, 2011 11:59 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Uni-trieve, antigen retrieval for immunofluorescence

Hello all,

Has anyone used the reagent 'Uni-trieve' from Innovex? It is purported
to
be a universal antigen retrieval solution that can be used at lower
temperatures (65-70 C for cytoplasmic antigens, and 75-80 C for nuclear
antigens):

http://innvx.com/unitrievepage.html

The company claims that the reagent is a universal retrieval solution
for
all antibodies and tissues (which is silly of course - how could they
know?). Does anyone have any experience with the product?

I am interested because I see that increased heat during antigen
retrieval
causes greater tissue autofluorescence during immunofluorescence
stainings. My current protocol is to use 20 minutes at 90-95 C on a hot
plate using sodium citrate buffer. Does anyone else either Uni-trieve or
a
different reagent/protocol for immunofluorescence stainings?

Thanks,

Andrea

Andrea Marion
Graduate Student
University of Illinois at Chicago


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[Histonet] Legal Case SOP

[Ann Angelo]

Can anyone share their SOP regarding the handling of legal cases (I.e. if a 
specimen is being requested from an outside source which is not the patient).  
Thank you, Ann


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[Histonet] Uni-trieve, antigen retrieval for immunofluorescence

[Andrea Marion]

Hello all,

Has anyone used the reagent 'Uni-trieve' from Innovex? It is purported to
be a universal antigen retrieval solution that can be used at lower
temperatures (65-70 C for cytoplasmic antigens, and 75-80 C for nuclear
antigens):

http://innvx.com/unitrievepage.html

The company claims that the reagent is a universal retrieval solution for
all antibodies and tissues (which is silly of course - how could they
know?). Does anyone have any experience with the product?

I am interested because I see that increased heat during antigen retrieval
causes greater tissue autofluorescence during immunofluorescence
stainings. My current protocol is to use 20 minutes at 90-95 C on a hot
plate using sodium citrate buffer. Does anyone else either Uni-trieve or a
different reagent/protocol for immunofluorescence stainings?

Thanks,

Andrea

Andrea Marion
Graduate Student
University of Illinois at Chicago


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[Histonet] Tissue handling at embedding

[Morken, Timothy]

What procedures do you have in place to prevent tissue loss at embedding? What 
do you do if tissue appears to have been lost?

And do you clean embedding molds before each reuse, or after one-days use 
(which may be many re-uses)?


Thanks for all info!


Tim Morken
Supervisor, Histology, IPOX
UC San Francisco Medical Center
Box 1656
1600 Divisidero St, B217
San Francisco, CA 94115
USA

415.514.6042 (office)
415.885.7409 Fax
tim.mor...@ucsfmedctr.org


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RE: [Histonet] How to store paraffin blocks ?

[Sherwood, Margaret]

We are a research group, but had paraffin blocks stored at room temperature for
20+ years.  We finally made the decision to just keep 2 years in the lab and
store 5 years off-site.  We trashed the rest.

Peggy 


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

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, September 20, 2011 10:20 AM
To: histonet@lists.utsouthwestern.edu; Itai Moshe
Subject: Re: [Histonet] How to store paraffin blocks ?

The usual procedure is to store at room temperature. It is assumed that the room
is air conditioned, otherwise if the temp is above 80ºF some blocks may stick
together, although that is not a real problem.
If at 4ºC you will need a refrigerated room or a series of refrigerators, which
is an additional and unnecessary expenditure.
We use to store the blocks for 9 years, unless it is a special case (used in
teaching) and those are stored forever.
Supposedly you use the slides for some procedure and those cut as "extras" are
kept along with the originals stained in the same files.
If you are referring to sections for IHC or other procedure, the epitopes will
be oxidized by the oxygen in the air in less than 2 weeks, so they can either be
stored in a fridge, or covered with melted paraffin, or stored in mineral oil.
René J.

--- On Tue, 9/20/11, Itai Moshe  wrote:


From: Itai Moshe 
Subject: [Histonet] How to store paraffin blocks ?
To: histonet@lists.utsouthwestern.edu
Date: Tuesday, September 20, 2011, 2:23 AM


Hi Histonet's,

What is the recommended way to store paraffin blocks for a long period ?
Room temperature, or 4C ?

The advantage in storing at 4C is that the blocks are always ready for
sectioning.
Is there a risk when storing at 4C that the humidity in the fridge will
cause damage to the paraffin blocks and the tissue ?
After sectioning, How do you store the slides, and for how long ?


Thank's
Itai Moshe
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Re: [Histonet] How to store paraffin blocks ?

[Rene J Buesa]

The usual procedure is to store at room temperature. It is assumed that the 
room is air conditioned, otherwise if the temp is above 80ºF some blocks may 
stick together, although that is not a real problem.
If at 4ºC you will need a refrigerated room or a series of refrigerators, which 
is an additional and unnecessary expenditure.
We use to store the blocks for 9 years, unless it is a special case (used in 
teaching) and those are stored forever.
Supposedly you use the slides for some procedure and those cut as "extras" are 
kept along with the originals stained in the same files.
If you are referring to sections for IHC or other procedure, the epitopes will 
be oxidized by the oxygen in the air in less than 2 weeks, so they can either 
be stored in a fridge, or covered with melted paraffin, or stored in mineral 
oil.
René J.

--- On Tue, 9/20/11, Itai Moshe  wrote:


From: Itai Moshe 
Subject: [Histonet] How to store paraffin blocks ?
To: histonet@lists.utsouthwestern.edu
Date: Tuesday, September 20, 2011, 2:23 AM


Hi Histonet's,

What is the recommended way to store paraffin blocks for a long period ?
Room temperature, or 4C ?

The advantage in storing at 4C is that the blocks are always ready for
sectioning.
Is there a risk when storing at 4C that the humidity in the fridge will
cause damage to the paraffin blocks and the tissue ?
After sectioning, How do you store the slides, and for how long ?


Thank's
Itai Moshe
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[Histonet] Verification System

[Ann Angelo]

I am looking for a verification system which does not require the data entry to 
be performed prior to cutting (ruling out Vantage and Cerebro).  I have looked 
at Thermo's slidemate but can't get them to move fast enough.
Does anyone know of another verification system that I may be able to try which 
does not require the data entry to be performed prior to microtomy?  Thanks, Ann



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