RE: [Histonet] hand processing rat brain-problem
It sounds like this tissue was immersion fixed, and the core was not reached by formaldehyde before considerable autolysis had set in. Or the perfusion was inadeqate to get formaldehyde everywhere. It definitely sounds like a perfusion/fixation issue. Do you know if the tissue was perfused, how long, what prewash, etc?
Consider the following link for a commercial product for reliable thorough perfusion.
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: firstname.lastname@example.org [mailto:email@example.com] On Behalf Of Danielle Crippen
Sent: Friday, August 06, 2004 11:14 AM
Subject: [Histonet] hand processing rat brain-problem
We are having a puzzling issue in paraffin processing rat brains from one particular investigator. We do all our paraffin processing by hand because we don't have the volume yet to require an automatic processor. Our standard protocol which has worked for every other tissue (rat brain, mouse brain, human brain, mouse embryo, bovine adrenal gland etc...) over the years is the following:
1.) Following fixation or perfusion (performed by the investigator or tissue bank--not us) with PFA or Bouin's: 30min-2hour wash in PBS
2.) 2x22 min 50% EtOH
3.) 2x22 min 70% EtOH
4.) 2x22 min 80% EtOH
5.) 2x22 min 90% EtOH
6.) 2x30 min 95% EtOH
7.) 2x60 min 100% EtOH
8.) 3x15min xylene
9.) 2x60 min in melted Paraplast Plus under vacuum (-50kPa = -15mmHg) at 600C
10.) 1x2h in melted Paraplast Plus under vacuum (-50kPa = -15mmHg) at 600C
When we followed this protocol with these particular rat brains, the center of the tissue (including ventricles, striatum, hippocampus....) completely fell apart while sectioning 7um coronal sections. The outer portion of the tissue (cortex etc.) seems very much intact.
We thought perhaps we had incomplete processing. The next time we received rat brains from this investigator we dissected for hippocampus (removing occipital lobe and cerebellum regions) in hopes this would increase the rate of infusion of all processing reagents. Then, we also left the tissue in paraffin under vacuum at 600C for 8hours with 3 changes, instead of 4 hours with 3 changes.
BUT the same thing happens when we section. Can it be a perfusion issue???
My apologies for the long winded message, but we are really perplexed!!
Many thanks in advance!!
Morphology Core Manager
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945
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