[Histonet] RE: need some help with frozen sections
C.M. van der Loos
c.m.vanderloos <@t> amc.uva.nl
Tue Apr 26 04:01:54 CDT 2005
Some DO's and DON'T's with immunostaining of cryostat tissue sections:
* 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: 2x 10 min 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 washbuffers surfactants may be
* 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
* 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,
I hope this isn't too much frightening you. Happy staining!
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
fax: +31 20 6960389
e-mail: c.m.vanderloos <@t> amc.uva.nl
----- Original Message -----
Eva C Anderson <eca9 <@t> georgetown.edu>
Mon, 25 Apr 2005 09:59:34 -0400
histonet <@t> lists.utsouthwestern.edu
[Histonet] need some help with frozen sections
I am starting to stain frozen sections for the first time. Until now I
have only stained paraffin embedded sections and so was hoping for
I contacted our histopathology lab who provided me with the following
They don't use any antigen retrieval method.
They pretreat the slides only with methanol for 10min followed by
Then they proceed with the normal steps of staining and dehydration.
I have however read that acetone can be used instead of the methanol.
What is the difference that this provides? I am trying to stain for
Is there anything els I should keep in mind when handling frozen
Would be very grateful for any information you could provide this
1. mailto:c.m.vanderloos <@t> amc.uva.nl
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