[Histonet] CNS Fixation

[Jan Shivers]

Sara,

This is what we do here with animal brain tissue:

1) Brain should fix for a 24-48 hrs in NBF.
2) They are trimmed in to about 5 mm thick (for embedding cassette).
3) Place cassettes face UP on the ice tray before sectioning (not face 
down).
3) The sections are cut at 4 um.
4) Waterbath temp is between 38-40. If they are exploding on the waterbath, 
then turn it down to about 34-36, making sure the wrinkles flatten out 
before sections are picked up.
5) Use adhesive slides.
6) For routine stains (H&E), the slides can either go in a 56C oven for 15 
minutes to get the water out or go directly onto the autostainer.  For IHC, 
they go into the oven at 80C for 30 minutes, then proceed with the IHC 
staining protocols.

Brains usually don't stay on if they are not fixed long enough, processed 
completely or have to much moisture in them from the ice tray.So, we don't 
put the brains face down on the ice tray. They are face up on the ice tray. 
This way they don't soak up water, they just get chilled.

Jan Shivers
Section Head
HIstology/IHC/EM
Univ. of Minnesota Veterinary Diagnostic Lab
St. Paul, MN


----- Original Message ----- 
From: "Breeden, Sara" <sbreeden <@t> nmda.nmsu.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, April 17, 2009 9:36 AM
Subject: [Histonet] CNS Fixation


Yes, I'm back.  I need the expertise of this group.



I am trying to convince my three pathologists that fixation in alcoholic
formalin is the best route for whole brain and spinal cord.  This has
been an uphill battle and in order to prevent sections from peeling off
during staining, I am still pre-drying necropsy brain slides for an
extra 20 minutes before putting on the auto-stainer (even this does not
prevent all instances). I need some expert ammunition for my "battle"
despite the fact that the SOP I've written requires it and it is rarely
followed.  If alcoholic formalin is not as good as some other method,
I'm open to suggestion. Of course, convincing them that thinner sections
would also help - but one step at a time!  Thank you in advance!



Sally Breeden, HT(ASCP)

NM Dept. of Agriculture

Veterinary Diagnostic Services

PO Box 4700

Albuquerque, NM  87106

505-841-2576



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