More on kidney frozen sections

From:Gayle Callis

-70C is still not cold enough, putting the biopsy into a -70C freezer is
not SNAP freezing, nothing will change in terms of section quality.     

You CANNOT prefix a kidney biopsy in alcohol and try to snap freeze it.
Alcohol acts as an antifreeze- retards freezing leading to poorly frozen
tissue, and really bad frozen sections plus it will probably mess up any
IFA.   There is a transport media often referred to as a fixative. It is
reversible i.e. must be washed out with its wash buffer in order to do snap
freezing. It is called Michels transport fixative, available commercially
in the USA, and could be made up in house.  However, the biopsy cannot be
left in this fixative indefinitely.  The recipe is found on Histonet (check
archives).  It can be held at room temperature during transport to your
lab, then you wash out the fixative with its wash buffer, and YOU snap
freeze (using Liquid N2 method) it for cryosectioning.  It is an excellent
transport media aka fixative for skin biopsies as well as kidney biopsies
destined for immunofluorescent staining.  A good way to handle biopsy when
there is a time delay.  

The bedside biopsy can be dropped into this media then transported
immediately to your lab for the freezing technic. I am reading your problem
as not being able to access liquid nitrogen in your lab easily?? Not having
access to liquid Nitrogen is a tough call - what has been described is a
commonly used, standard method for snap freezing kidney biopsies. 

You are trying to cool down a lot of metal by freezing the tissue onto that
metal holder. It would be better to use a small plastic embedding mold,
freeze tissue in this small mold, then mount block onto a metal chuck.
This way the tissue will freeze even faster.  One can also snap freeze
using a coverslip, a drop of OCT, then add tissue, freeze, warm up back of
coverslip by rubbing it to release block, mount block onto metal chuck.  (A
Peggy Wenk trick that really works!) 


I forget to mentioned that we cut 4um frozen sections.And as liquid nitrogen 
is not very convenient for the technician in our routine work.I just wonder 
if we could put the OCT-specimen in a -70C fridge for quick freezing?

And could I do as below,
Fix the fresh specimen in a kind of fixation solution first(like ethanol) 
and then transfer it to the lab at 4C.
Immerse the specimen in OCT on a metal holder.
Put them all in -70C immediately.
After OCT is frozen,transfer them quickly into the cyrostat which adjust to 
-18C and then sectioning.
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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