Re: cryostat, questions

From:Gayle Callis

At 01:33 PM 7/2/2003 -0400, you wrote:
>Hi Histonetters,   I have a few questions about cryostat sectioning.    

In what situations is a cryostat better than regular paraffin processing,
and why?  

Lipid staining, enzyme histochemistry  and immunohistochemical staining
where formalin fixation, acid decalcification or paraffin processing i.e.
solvents and temperature compromises either the enzyme or antigen (the
latter when no retrieval ever works - murine CD4 and CD8 are examples.) 

How do you get cryostat sections to stick to a slide for staining? 
Air dry 30 minutes or so after cryosectioning using Plus charge or Gold
Plus slides with formalin fixed, sucrose cryoprotected tissue,  Unfixed,
fresh frozen sections are air dried anywhere from 30 min to overnight for
immunostaining.  For rapid H&E, immerse immediately into NBF, rinse and

Do you need to fix tissue for cryostat in paraformaldhyde/sucrose solution? 
No, if you do this, fix first then cryoprotect overnight in 20% sucrose,
let sample sink to bottom of container. We never combine these solutions.  

Can you section fresh/frozen tissue?  
The tissue must be SNAP frozen, not just frozen in a cryostat - that is too
warm for immunostaining work. Answer is yes, even thin 2 um thick sections
using good disposable blades. 

In our lab, we exclusively do more frozen sections than paraffin sections
for immunohistochemistry for murine IHC/IFA (fluorescent) work. WE also do
FS for ISH, and have seen it used exclusively for Laser Capture Dissection


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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