[Histonet] CAVEAT on Methanol for acetone/alcohol fixation for murine CD IHC, RE: need help on frozen sections,

Gayle Callis gcallis <@t> montana.edu
Tue Apr 26 12:45:19 CDT 2005

Chris's comments are also true for MURINE CD marker 
staining.  Consequently, we avoid methanol for both fixation and peroxidase 
blocking on murine frozen sections for CD (lymphocyte) IHC staining.

******Caveat:  We NEVER use methanol as a frozen section fixative for 
MURINE lymphocyte (CD) markers.  The acetone/alcohol (AA) fixative we use 
with great success is a mixture of  acetone and 100% ETHYL ALCOHOL, and 
NEVER Methanol.  Overnight air dried frozen sections are fixed in: 75 mls 
ACS reagent grade acetone and 25 ml 100% ethanol for 5 minutes at 
RT.  Sections are NEVER allowed to dry after this fixation but go directly 
to buffer, 3 changes.  It obviously has worked well for us as we have one 
CD4 protocol where rat antiMouse CD4 monoclonal is diluted 1:15,000 (fading 
out at 1:20,000) of a 0.5 mg/ml concentration.

#####Please note:  This combination "AA " fixative is used for MURINE CD 
marker staining for frozen sections and NOT human CD markers.  I defer to 
Chris's experience on human CD marker staining, as the ethanol component in 
AA will probably cause failure with human CD marker also.   We are talking 
two very different species here.

Chris wrote:
>   Some DO's and DON'T's with immunostaining of cryostat tissue sections:
>           * The  application  of methanol, either as a fixative or as 
> base for endogenous   peroxidase   activity  blocking  is 
> (at  least       to my vision) absolutely  excluded.  For  example, most 
> human CD markers are completely destroyed by methanol.On 
> the           other hand I am aware of investigators  who  are  using 
> successfully an       acetone/methanol mixture for fixation of 
> mouse     cryo's for staining CD4, CD8 etc.

I have talked about methanolic bridges in the past which may cause loss of 
CD marker staining, but maybe this is NOT the exact mechanism for MEOH 
damage to antigens.  Jules Elias indicated in his book that it was "thought 
that immunoreactivity of intracellular antigens may be affected by MEOH 
destructive effects on surface membrane antigens."  He never said what the 
action was of MEOH on surface markers, but this came to me by way of 
discussion with a knowledgable person. Biogenex, in some printed material, 
also indicated MEOH should be avoided for CD marker work.  It would be 
interesting to pursue what MEOH actually does to CD surface marker???? Any 
takers on the chemistry of what is going on here??

Elias supported his statements with the following references from his 
book:  Elias JM  Immunohistopathology, a practical approach to 
diagnosis.  First Edition ASCP press, p46 - 48.

1. McMillan et al.  J Cutan Pathol 8:228, 1981.  Demonstration in situ of T 
cells and T cell susets in lichen planus using monoclonal antibodies.

2. Matsumoto Y.  simultaneous inhibition of endogenous avidin binding 
activity and peroxidase applicable for eht avidin biotin system using 
monoclonal antibodies.  Histochemistry 83:325,1985

3. Van Duijin P.  Histochemistry of DOPA factores: III. Inactivation 
experiments on the DOPA factores in neutrophilic and eosinophilic 
leucocytes and erythrocytes.  Acta Physio. Pharmacol 5:428:1987.

Recommendations from the experts (Chris van der Loos) are well taken in 
order to save time, reagents, and avoid frustrations.
For certain murine CD IHC staining on frozen sections,  we discovered that 
acetone fixation recommendations from the antibody manufacturers were often 
far less successful than using the acetone/ethanol mixture, a method 
graciously given to us from an murine IHC expert in the field.

Now we do a fixative panel to determine what works best for any given 

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

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