[Histonet] sections lifting along the edges

[Patsy Ruegg]

I would fix the profused fixed tissue in formalin for 12-24 hours before
infiltrating it in 30% sucrose then snap freeze it, the researcher who
thinks it is over fixed is wrong and that is very common, researchers think
that aldehyde fixations inhibitis IHc staining but things have changed and
especially with antigen retrieval techniques it is better to fix and then
infiltrate in sucrose and snap freeze to get the best results, profuse
fixation for 10 min is not enough.

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tyrone
Genade
Sent: Saturday, November 05, 2011 3:03 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] sections lifting along the edges

Hello,

I have a problem with sections which lift off the slides. The slides
are APTES coated and it isn't the entire section lifting off the
slide, just the peripheral bits.

I did not prepare the tissue nor section it. I am told the rat was
killed and perfused with PFA for 10 minutes and then the brain removed
and dehydrated in sucrose. It was NOT postfixed overnight in PFA. The
dehydrated brain was then set in tissue tech and sectioned on a
cryostat. 40 micron sections were made.

These sections are lifting along the periphery. What is more is that
antibody staining is not consistent through the sections and antibody
penetration is only about 4 microns.

Various conflicting reasons are given for this lifting and antibody
inconsistency problem. The histologist suspects that the tissue was
either not fixed enough or the the sections were not left to adhere to
the APTES slide for long enough before being frozen at -20 oC. The one
prof things the tissue was over-fixed.

One of the antibodies we are using is the cannabinoid receptor CB1
antibody. Our signal is really poor and bleach very quickly. We found
an article where the tissue was postfixed (presumably in PFA) over
night and the staining is excellent...

The person who did the section reported that she saw what looked like
ice-crystals in the tissue. I wonder if this could be a sign that the
fixation was not homogenous, i.e. the tissue now had a different
texture which made it look like ice crystals. I also wonder about the
-80 oC freezer which was used. It is very full and so probably serving
more as an insulator than a rapid freezer. As such freezing may have
been slow and ice-crystals could have formed but I don't really see
tissue damage typical of freezing and the sections life along the
periphery not the center...

Any ideas on what is happening would be much appreciated. The person I
am working with still has rats to kill and we would like to be sure
that their brains will be fixed properly and not go to waste.

Thanks
-- 
Tyrone Genade
http://tgenade.freeshell.org
email: tgenade <@t> gmail.com
tel: +27-84-632-1925 (c)
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