[Histonet] GM-CSF in mouse tissue, or secreted proteins in general

Wed Jan 11 12:53:10 CST 2006

Hi all, 
I was wondering if anyone has ever successfully detected GM-CSF secretion in mouse tissues using immunofluorescence? (NOT on paraffin embedded sections- I have seen those papers).
Do various tissues express this cytokine at different levels?

Also, do secreted proteins have to be fixed and go through the whole paraffin embedding process to be properly preserved and then subsequently detected? Just curious. I always seem to find sources of staining only using chromogenic techniques on paraffin sections and the staining always seems a little suspect, kind of difficult to interpret what is background and what is real. 
Thanks for any input.
Melissa

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Today's Topics:

   1. Methanol and CD markers  (Gayle Callis)
   2. Re: carbowax (Gayle Callis)
   3. TBS Polyfin (Steven Coakley)

----------------------------------------------------------------------

Message: 1
Date: Wed, 11 Jan 2006 08:42:11 -0700
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: [Histonet] Methanol and CD markers 
To: Lori Richey <lrichey <@t> u.washington.edu>,
	Histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20060111083916.01b50598 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Just a note here. Methanol is known to create problems with CD marker 
immunohistochemistry and that is why many use hydrogen peroxide in buffer 
instead of this solvent.   This point was brought up in Jules Elias's book 
on immunohistochemistry and also on Histonet in the past. (see archives).

At 04:02 PM 1/10/2006, you wrote:
>We use both CD4 and CD8 on FFPE sections.  We had problems with the CD4 
>until we changed our quenching step to 0.5% H2O2 in MEOH for 10 min. The 
>pretreatment for both is 15 min microwave in pH 8 EDTA.
>
>Thomas Pier wrote:
>
>>Hello,
>>I am looking for Reinhard von Wasielewski.  I recently saw a post
>>stating that he had working protocols for CD4 and CD8 that even worked
>>in decalcified bone marrow.  CD4 and CD8 in FFPE, decalcified bone
>>marrow is exactly what I'm having problems with.  I have been banging my
>>head against the wall with this for some time now.  I would really
>>appreciate any help I could get on this.  Thank you very much.
>>
>>Tom Pier
>>
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

------------------------------

Message: 2
Date: Wed, 11 Jan 2006 08:44:21 -0700
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: Re: [Histonet] carbowax
To: Erin Wrona <ewrona <@t> yahoo.com>, Histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20060111084247.01b63270 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Material safety data sheets should give this information as would a Merck 
index.  Sometimes companies like Aldrich have this in their product 
description in their catalog too or EMS will tell you via tech services.

  At 05:52 PM 1/10/2006, you wrote:
>Hi all,
>
>   Our lab director wants us to try using carbowax polyethylene glycol as 
> an embedding medium because it has a slightly higher melting point than 
> our paraffin. I have never used it other than in formulations for frozen 
> sections, in which case it is water soluble.  One of the other techs here 
> thinks that it is always water soluble.  Does anyone know for sure?
>
>   The specifics are:  ems product #19770, PEG 8000 flakes, melting point 
> 60-63 degrees.
>
>   T

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

------------------------------

Message: 3
Date: Wed, 11 Jan 2006 08:01:44 -0800 (PST)
From: Steven Coakley <sjchtascp <@t> yahoo.com>
Subject: [Histonet] TBS Polyfin
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID: <20060111160144.83423.qmail <@t> web90205.mail.scd.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Has anyone ever used the TBS Polyfin paraffin for tissue infiltration and embedding.  I'm considering a change from the paraplast I currently use.

  Steve

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