[Histonet] immunostaining unfixed frozen sections- long email

Tue Feb 6 17:29:42 CST 2007

Dear Sonya and Teri,

Teri, yes I have done this, but is tricky to keep section on the slide and 
was a bit tedious with the fear of losing the section.   We prefer to do 
either acetone or acetone alcohol fixation of an overnight air dried frozen 
section, then and a standard immunofluorescence staining with primary and a 
secondary conjugated to a fluorophore.

  As for a direct FITC conjugated primary antibody, you may have problems 
if the FITC quenches itself.  This happens with CD4-FITC antibodies (also 
CD8-FITC), The problem is caused by the FITC molecule in close proximity to 
other adjacent FITC molecules ( as my physical chemist hubby explained) the 
FITC quenches.  This is NOT photobleaching, which is cause by exposure of 
fluorophores to room light and/or UV excitation light during 
microscopy.  For unfixed frozen section staining, I worked with a purified 
CD4 antibody at a very high concentration 3 ug/ml (do a dilution panel), 
then after staining, fixed with formalin or paraformaldehyde and come back 
after fixation with a secondary antibody.  You could try a direct FITC 
conjugated primary, then fix, rinse and mount a coverslip.  If you get no 
staining, you may want to consider the quenching problem.

Now for unfixed frozen section immunostaining particulars

Frozen section mounted on Plus charge slide.  This may be the time to try 
the Plus Gold slides which are touted to hold frozen sections better.  Air 
dry the frozen section (overnight at RT should work).  Draw lines across 
slide above and below the section so you can add 100 ul of any 
reagent,  ImmEdge hydrophobic barrier pens from Vector are excellent, and 
it is easier to blot from a corner rather than a circles edge.
CD4 antibody (not a FITC conjugate) was at 3 ug/ml concentration.   Diluent 
for CD4 was 5 to 10% goat/2.5% mouse serum and is the normal serum 
block.   You can try a normal serum block and in this case it would have 
been the 5 to 10% goat serum with 2.5% mouse, change the normal serum, we 
do not use super blocks for this protocol

Primary incubations are done at 4C, and the buffer contained normal serum 
(0.1 to 0.2% matched to host of secondary or goat serum).  We avoided 
detergent due to the delicate frozen section.  If you want to do an 
overnight incubation or enzyme method after fixation, you need to add 
sodium azide, 0.1%  to retard any bacterial growth.  Azide also retards 
endogenous peroxidase.

Protocol

1.  Lay slides flat in humidity chamber
2.  Add buffer with a plastic Pasteur pipette introduced from side of 
section rather than squirt on top of section - very slow 
flow.  This      will rinse away OCT, let stand approx 1 minute then pick 
up slide, drain it onto a towel, blot from corner.  Work one slide at 
a        time.

3.  Add normal serum block in same manner, incubate at 4C for 20 min or so
4.  Blot off normal serum block, blot from corner, don't rinse!
5.  Add primary antibody and incubate for 30 to 60 minutes (you will have 
to do a time study to see what is best for your antibody)
6.  Drain off antibody,  add rinse buffer gently for a minute or so, drain 
off and repeat.   Don't use coplin jar rinsing, this is too  vigorous.
7.  Fix in 2% NBF by immersing slides into fixative for 8 to 10 min.  This 
is diluted NBF or you can make it up this way.

If you plan to do HRP method after fixation, you could try a gentle say 
0.03% H2O2 in buffer or do AP method to avoid this block. Do not use a 
methanol peroxidase block, opt for buffer/H2O2 instead.

8.  After fixation, resume a normal immunostaining protocol by adding a 
secondary conjugated to a fluorophore or even enzyme or biotin, then 
proceed with Strepavidin-HRP, etc.  The fellow who taught me this used 
Vector ABC kit.  If you use this, I suggest using an F(ab')2 secondary and 
if we used a fluorophore conjugated secondary antibody it was F(ab')2 frag 
of IgG and adsorbed to mouse.

You will have to adjust your antibody concentrations, maybe even the 
secondary antibody.  Good luck

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

Sonya,

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
(room
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
specificity.

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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Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717