Re: Harvesting tissue Sub P and CGRP

From:Gayle Callis

You didn't say how you are staining, assuming immunohistochemistry (IHC)? I
assume you have endogenous peroxidase present during staining, correct?
You didn't say how you do your IHC protocol  

There are ways to quench all peroxidases in blood when using peroxidase
method for IHC.  Our snap frozen sections of mouse spinal cord (not
perfused) do not show endogenous peroxidase staining after HRP method after
good endog peroxidase blocking - mouse cord being a smaller version of the
rat cord with same blood problems.   

If you use standard endogenous peroxidase blocking method with methanol on
frozen sections, you may chew up sections plus pseudoperoxidases may not be
totally quenched.  Been there and had this happen using DAKO peroxidase
block for frozen sections.  There is the glucose oxidase method the
quenches all endog peroxidases and pseudoperoxidases, it is wonderful for
frozen sections and works with FFPE also.  I would be happy to send it to
you via private email. 

We remove cord, embed in OCT inside Tissue Tek cryomolds after orientation
for either midsagittal or transverse (cross sections,) then snap freeze
with dry ice/isopentane slurry - whatever works best for you. 
One can embed cord laying flat for mid sagittal sectioning, and turn block
and mount on chuck so cord is on end - we end up grooming the block by
adding more OCT around original block to build up a good cutting face. So
many ways to play. 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


TheAt 08:56 AM 8/15/2003 -0500, you wrote:
>           We will be harvesting spinal cord that will be reacted with
>Substance P and CGRP. We are unable to perfuse the rat due to a portion of
>the animal will have to be placed in a beetle colony. After the cord is
>removed should I immersion fix it or snap freeze it with liquid nitrogen?
>When reviewing the sections will I have a problem seeing my reacted areas
>due to blood in the tissue since I am not perfusing?  Thank you,  Clover
>Daley  Palmer Center for Chiropractic Research       

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