[Histonet] sections lifting along the edges

[Kim Donadio]

Seems to me if you can fix the sections on the slides before staining that might help. However, Ive seen when using thick sections as this is where the periphery of the tissue meets the slide creates a gradient where the reagents incounter the most resistance, causing the section to peel away(also a more gentle method?)(heat?). As far as the low antibody sensativity, perhaps you could try longer incubation. I found a couple of articles on this subject that might help. Also, every article I have seen on this says to store your antibody at-20C . We always stored our antibodies at -60C so I am not sure if this is something specific to this particular antibody. Here's the articles. Hope this helps. 
 
http://www.ihcworld.com/smf/index.php?topic=6257.0
 
http://www.labome.com/product/Thermo-Scientific-Pierce-Products/PA1-745.html
 
This next article actually has a IHC and IF method for this antibody
 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2324193/ 
 
Kim
 
 
 
 

________________________________
From: Tyrone Genade <tgenade <@t> gmail.com>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Saturday, November 5, 2011 5:02 AM
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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