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