[Histonet] RE: Staining frozen sections without fixation
TJJ <@t> Stowers-Institute.org
Tue Feb 6 12:46:46 CST 2007
You wrote: >>I'm trying to get an aPDCA-1 antibody to work on frozen
mouse spleen sections. The antibody is normally used for FACS however
the company says that it works for immunhistochemistry. They recommend
staining before fixation. Usually I cut frozen sections, dry overnight
temp) and fix in acetone (10min at room temp) before putting the
sections in PBS and continuing with staining procedure.
If I stain before fixation;
Will the sections survive?
When should I fix - after primary, secondary etc?
What should I fix with?<<
Have you tried it using acetone fixed frozen sections yet? If so, did
you get any staining? You might need to use the antibody at a higher
concentration than what you are accustomed to using for IHC.
Are you using conjugated primary antibody? What detection method are you
using? If you are using a rat anti-mouse PDCA-1, be sure to use a
mouse-adsorbed anti-rat secondary antibody for better species
It might be interesting to try doing whole mount incubation of say a
FITC-labeled antibody in mouse spleen, overnight at 4 degrees C, rinses
in PBS or TBS, then brief formalin fixation followed by sucrose
cryoprotection**, freezing, and then sectioning. Use an anti-FITC AP
(not HRP, so you don't have to worry about quenching endogenous
peroxidase) or anti-FITC Alexa 488 for fluorescent detection. You can
also try doing the entire immuno procedure on whole mount prior to
fixation. Alkaline phosphatase activity and some fluorescence is
maintained after formalin fixation and cryosectioning. Remember to run a
negative control spleen sample in parallel with non-immune serum for the
primary antibody incubation.
**I believe with the immersion of the tissue in the buffer for an
extended period of time (overnight), if you do not fix and cryoprotect
you will see lots of freezing artifact due to the buffers soaking up
into the tissue.
I've never done anything like this before, so all I can offer are
suggestions and a hearty "good luck!". Hopefully Gayle Callis will chime
in with some other suggestions. It wouldn't surprise me to hear she's
done something like this successfully in the past.
FWIW, I'm not usually very optimistic when trying an antibody optimized
for FACS in IHC.
Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110
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