Are you working with human brain sections? What size? The two days
fixation, is that whole brains or dissected sections? I agree with the
recommendation by Joyce that you should air dry the slides (make sure no
water underneath sections) BEFORE placing on the hot plate. It will not
only help section adherence, but will help to prevent nuclear bubbling
from the water "cooking" under the section.
It is entirely possible that you have a fixation problem. I've found
when we rush fixation on autopsy brain sections, they may cut with no
difficulty. Then as we stain (frequently in the bluing stage) they float
off the adhesive slides right before my eyes!
All of this may be contributory to animal work as well, but I know I've
experienced it with human tissue, first hand. As for your requests for
staining techniques, I'd be glad to forward our amyloid methods
separately. We typically do not to Iron stains here in Neuropathology,
but there are many good protocols out there for Perl's.
Sorry to ask so many questions in response to yours. Feel free to
contact me if I can be of further help.
Have a great rest of the week.
Jennifer L. Hofecker, HT(ASCP)
Vanderbilt University Medical Center
Division of Neuropathology
NSH Quality Control Committee Chair
[mailto:firstname.lastname@example.org] On Behalf Of Ryan
Sent: Friday, February 08, 2008 2:58 AM
Subject: [Histonet] brain tissue sections
Please help me with my brain tissue sections, I'm having difficulties in
staining them because they disintegrate during H&E staining, resulting
into folded and incomplete sections into the slide.
I processed the tissues 2 days after fixation using Leica ASP300S (all
new reagents). I have no problem in cutting 5 u thickness during
microtomy. I used adhesive pre-treated slides and Milli-Q water during
orientation and fishing out in the floatation bath. I use flattening
table as hot plate and heat the freshly cut slides at 62C, for not less
than 10 minutes. During staining, I use 3 changes of xylene and abs.
ethanol followed by 80% then 70% ethanol, all of them for 3 minutes. I
used milli-Q water for hydration and washing during staining. For
differentiation and bluing, I use 1% acid alcohol and ammonia water.
Can you also provide me some techniques and difficulties for Perl's
prussian blue and amyloid staining.
Thank you very much for your help and your website which changes the
lives of ordinary histologist like me.
More power and God bless!
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