RE: IHC on frozen cryosectioned rat kidney

From:"Stone, Jessica" <>

No problem:
I snap froze the kidney two ways; the first was directly submerging in
liquid nitrogen, the second was I put the piece of kidney in a plastic mold
filled with OCT and then submerged this in isopentane chilled with liquid
nitrogen. The first method I got good signal but poor morphology so with the
second method (tried two different times) I was trying to improve morphology
but, I got no signal... 

As for after sectioning, I let them sit at room temperature approx. 5-30 min
(time of sectioning), then into Zn-formalin at RT for 5-10 min., rinse Zn
with water, then wash PBS, incubate donkey serum block (actually the first
run was blocked with BSA, don't think this is the critical factor...?), wash
PBS, then primary dilution series (non-commercial rabbit polyclonal to a
Hepatic Nuclear Factor), PBS wash, then Cy3 conjugated donkey anti-rabbit
from Jackson, PBS wash, lastly DAPI-- which always stained great in all
cases. Also, I run liver each time as positive control for this antigen, and
it worked in all cases.

I also have tried overnight fixation in Zn-formalin followed by processing: 
water 15'
100% prosoft dehydrant 1 h
100% prosoft dehydrant 1 h
100% prosoft dehydrant 1 h
100% prosoft dehydrant 1 h
Clearant			1 h
Clearant			1 h
Clearant			1 h
Clearant			1 h
paraffin wax 58'C        45 min
paraffin wax 58'C        45 min
paraffin wax 58'C        45 min
paraffin wax 58'C        45 min

this is a processing schedule left by the prior histotech... 
I got no signal with this method, tried proteinase K digest and HIER--I had
problems though...

I have been struggling with this for a couple months and finally decided to
put my dilemma out to this group. 

Thank you for any help, ideas, comments, criticisms...

-----Original Message-----
From: Gayle Callis []
Sent: Tuesday, May 22, 2001 7:47 AM
To: Stone, Jessica
Subject: Re: IHC on frozen cryosectioned rat kidney

More details please.  How do you snap freeze the kidney?  and what do you
do to section after picking up on slide?  What is your antibody?  and how
do you do your staining?  Lots of questions, but easier to give suggestions
if specifics come forth.

At 01:10 PM 5/21/01 -0700, you wrote:
>If you have time and are so inclined, can you write me the best (or
>successful methods in your hands) for initial freezing of tissue (rat
>~5mm thick); and post cryo-section processing for immunohistochemistry
>(doing indirect with Cy3).  I am a beginner and having problems with cell
>damage and inconsistent results; I believe culprit is initial freezing
>method or tissue treatment after sectioning i.e. dry or not to dry
>if dry then before and/or after fixation, best fixative and time in
>fixative, does the tissue need to be perfused etc. etc.
>Thank You for your time and help!!
>P.S. any thoughts on switching to a  fixed, paraffin embedded method?? I
>tried it once and got no signal, not sure if overfixed, overprocessed, or
>Cy3 bad choice...
>Jessica Stone
>Ass. Scientist
>Ligand Pharmaceuticals
>La Jolla, CA
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

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