Some
Department of Pathology
Academical Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
After cutting, let the sections dry overnight at room temperature under a ventilator.
Fix with cold acetone (10 min, 4C). Some people apply a "double
acetone fixation" method: 2x10 minutes acetone fixation with
air-drying in between. This may improve the tissue morphology.
The quality of the acetone should be
P.A. grade and don't use it twice. Re-distilled acetone cannot be
used for fixation of cryo's. Traces of water in the acetone ruins
the tissue morphology.
Be aware that acetone is not a real
fixative like NBF. Acetone just solves the fatty membranes and
coagulate the proteins. Cryostat tissue sections remain quite
vulnerable with respect to surfactants like Triton-X100, Tween-20.
These should be avoided. Be aware that in some autostainer wash
buffers surfactants may be included.
To solve this problem of tissue
vulnerability an extra fixation (after acetone-fixation and
air-drying) with Zamboni's (1 min, RT) is optional. This extra
fixation step also improves the tissue morphology. However,
depending on the antigen to be IHC stained, this step may also
decrease the IHC staining intensity.
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.
Acetone is not a good fixative when
staining nuclear antigens. This fixation will lead to quite fuzzy
stained nuclei. Instead, a NBF-fixation (5 min, RT) works out well.
Although NBF-fixation is used: do NOT apply heat-induced antigen
retrieval.
Some antigens associated with fatty structures (for example, oxLDL, or apoB) will solve into acetone. In this case NBF (5 min) can be tested. Again, NO antigen retrieval!
Endogenous peroxidase activity can be blocked with 0.1% Na-azide +
0.3% peroxide in PBS or TBS (20 min, RT). This kills at least the
endogenous peroxidase activity in erythrocytes effectively, but in
neutrophils some will remain. If endogenous peroxidase activity of
neutrophils is a real problem, glucose oxidase blocking method is
optional (see Histonet archives).
At the end of the IHC staining procedure, after the chromogen step wash your slides with tap water. NEVER use distilled water as this will ruin the tissue section completely!!!!
Unlike with FFPE sections, aqueous solutions (depending on the
chromogen used of course) is no problem for mounting cryostat tissue
sections.
The concept of first cutting a tissue
section and than fixation means "post-fixation". This is something
different from a FFPE section that is first fixed as a block, than
embedded and cut
("pre-fixation"). This means that soluble antigens may leak away
from a "post-fixed" tissue section. Be aware of this when IHC
staining any small protein (<50KD), cytokine, chemokine, hormone,
etc.
References:
1. McMillan EM, Martin D, Wasik R, Everett MA (1981) Demonstration in situ of "T" cells and "T" cell subsets in lichen planus using monoclonal antibodies. J Cutan Pathol. 8(3):228-34. PubMed Abstract
2. Matsumoto Y (1985) Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies. Histochemistry. 1985;83(4):325-30. PubMed Abstract