Re: [Histonet] Frozen cell pellet

From:Gayle Callis

How many cells you spin down may be critical.  Here is a protocol that 
works for us.

1.  1 to 3 x 10 (to the 7th) cells/ml.
2.  Spin down and rinse three time with PBS.
3.  Add OCT (approx 1 ml or less) to last pellet, mix.
4.  Immerse tube directly into liquid N2.
5.  Pop block out with a sharp rap inside cryostat.

Mount block and section.

The key is to make sure you don't have too many cells and do a good mix 
with OCT in order to disperse any water from buffer and spread out 
cells.  We have sectioned thinner, 4 um rather than 5 to insure uncrowded 

We build block up with more OCT around end of block so the block face is 
not so tiny and you can manipulate the tiny section.  Chris van der Loos is 
a master at the supertiny section!!

A wider conical end microcentrifuge tube (holds 2 ml or more overall) 
spreads things out a bit more for a broader faced block but a 1.5 ml tube 
will work.
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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