RE: IHC on frozen cryosectioned rat kidney

From:"Stone, Jessica" <JStone@ligand.com>

Thank you for your input; I'll try the isopentane/dry ice slurry method
you've describe, and then some different fixation methods etc. Since, it did
work on the frozen liver, I'm hopeful I'll be able to get it in the kidney
with some methodical troubleshooting... Actually, I haven't had it work in
kidney or liver via the fixation-paraffin method. I was going to try a
shorter fixation-processing schedule and then a DAB reaction instead of the
fluorescence...

Well, here I go...

thanks again
Jessie

-----Original Message-----
From: Gayle Callis [mailto:uvsgc@montana.edu]
Sent: Tuesday, May 22, 2001 3:17 PM
To: Stone, Jessica
Subject: RE: IHC on frozen cryosectioned rat kidney


I know one gal who always soaked her fresh tissue in diluted OCT (dilute
with PBS) for a few minutes, then used a Peel away Mold to snap freeze in a
hexane/dry ice slurry, rather than isopentane/dry ice slurry.  She did
embed in OCT after the soak, you could line them up, then freeze, the lag
time will let them "cryoprotect" a tidge.  Hexane gives a slightly slower
freeze, but it works as well as isopentane/dry ice slurry, and is the
method of choice for bone.  

I prefer Tissue Tek cryomolds, ONLY, they have thinner plastic and less
time for freeze.  I embed in mold, and slowly lower bottom in until it
turns white, then lower into dry ice isopentane slurry.  This also works
with hexane.  Hope you have a hood, bad stuff!! to breathe!!  I gave up on
liq Nitrogen years ago, it was a mess - always having to recool the stuff.
Ugh! 

Your kidney seems the right size, and animal tissue has less fat than human
tissue, I think the classic method for snap freeze of human kidney biopsy
may not work as well as for animal.   

Don't know what to say on antibody staining if it works on your snap frozen
or fixed liver, then it should work on kidney as long as you have antibody
optimized along with technic.  At a loss there.


At 02:02 PM 5/22/01 -0700, you wrote:
>Thank you for your time and input. I'll definitely try out some of your
>ideas. Also, I believe in the original e-mail is where I indicated tissue
>size, I cut out a kidney midsection ~5-8mm thick.
>Thanks again
>Jessi
>
>-----Original Message-----
>From: Gayle Callis [mailto:uvsgc@montana.edu]
>Sent: Tuesday, May 22, 2001 10:41 AM
>To: Stone, Jessica; histonet@pathology.swmed.edu
>Subject: RE: IHC on frozen cryosectioned rat kidney
>
>
>Trying to snap freeze a whole rat kidney, if that is the case, may be too
>much tissue for a good snap freeze.  Have you tried a smaller sample,
>bisected kidney or portion of a kidney?  I am assuming the kidney is whole
>but I may be incorrect, you did not say??  
>
>We snap freeze all murine and rat tissues embedded in OCT in a Tissue Tek
>cryomold with little plastic wing edges removed for a better heat sink, and
>immerse this into dry ice cooled isopentane until bubbling stops.  The dry
>ice and isopentane are mixed together giving a slurry, and tissue would be
>cut at -20C.  I do whole mouse kidney, smaller than rat, this way and have
>no freezing artifact, kidney looks normal.  
>
>After sectioning, sections are air dried 30 min before acetone or air dried
>overnight before acetone alcohol mixture, you would have to optimize a
>fixative protocol for your antibody, some are fussier than others.  NBF or
>paraformaldehyde fixation can be done more rapidly, but for a shorter
>fixation time to prevent overcrosslinking.  I have fixed as little as 2 min
>in 2 -4% paraformaldehyde without losing sections, but I did not do any
>retrieval either.  Acetone 75%/100% ethanol 25% 5 min RT then into PBS
>gives better morphology than acetone 10 min at 4C.  It must be pointed out
>I am not using your antibody for my work.  
>
>I have no idea if this helps, just tossing it out.
>  
>
>Gayle Callis
>MT,HT,HTL(ASCP)
>Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>Bozeman MT 59717-3610
>
>406 994-6367
>404 994-4303 (FAX)
>
>
Gayle Callis
MT,HT,HTL(ASCP)
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)



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