[Histonet] tissue separating from OCT


Hello Histonetters:

I am having difficulty sectioning this mouse brain.  The tissue is separating
from OCT upon sectioning.  Let me give you a brief background of how the brain
was processed.  Perfused with 4%PFA, fixed overnight at 4C(same fix), next day
rinsed with 1XPBS, then serially cryoproctected overnight with 20%, 30%, 40%
sucrose/1XPBS - tissue sunk every time.  Rinsed tissue from the sucrose with
1XPBS, blot dry, then flash froze in OCT, immersed in isopentane @ -80C. I am
trying to section at 10um.  I've done this procedure before, many, many times,
never had problem trying to get a nice section.  I am about to pull my hair
out!!!  What's the point in putting a tissue that has wrinkles on a slide.  I
don't know what my problem is!    I've adjusted the angle on the cryostat &
adjusted the temperature, changed blades, changed the position of the tissue to
make almost a full circle(cryostat specimen holder).  Can someone in histoworld
help with this frustration?  I thought I was just having a bad cutting day, but
this is now carried on for over a week.  I need to section the entire brain -
ever piece is precious to me.



Dina D. Gaupp, B.S., M.T.
Medical Research Specialist
Center for Gene Therapy
Tulane University Health Sciences Center
JBJ Bldg/Rm 658, SL-99
1430 Tulane Avenue
New Orleans, LA 70112
Lab: 504.988.1194
Fax: 504.988.7710
Email: dgaupp@tulane.edu

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