[Histonet] holly nuclei bat man

[Patsy Ruegg]

Yes this all makes good sense and explains why I may not be able to have
control of this because I do not always do the tissue fixation and
processing myself.  That first step of adequately fixing (cross linking the
proteins) to protect them from tissue processing and AR are sooooo
important.  The one thing I can control is to make sure the slides are
completely dry before baking them as Renee mentioned.

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting, LLC
40864 Arkansas Ave
Bennett, CO 80102
Fax: 720-859-4110
pruegg <@t> ihctech.net
-----Original Message-----
From: Gudrun Lang [mailto:gu.lang <@t> gmx.at] 
Sent: Sunday, January 06, 2013 4:32 AM
To: 'Patsy Ruegg'
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: AW: [Histonet] holly nuclei bat man

I have a quiet semi-scientific explanation. Formalin-fixation stabilizes
proteins via crosslinking. AR "solves" methylol-adducts due to formaldehyde
and perhaps also some crosslinks. Nuclei also have a protein-scaffold,
filled with nucleinacids. It seems like a "ladder" or "hole in tights". It
seems like there is a kind of tension, after breaking a crosslink the hole
"blubbs" out.
This is also seen with pretreatment in hybridization-procedures. The more
enzymatic and heat-retrieval the more holes.  
And there is a direct relationsship between fixation-quality and
AR-strength.

Bye Gudrun

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Patsy
Ruegg
Gesendet: Samstag, 05. Jänner 2013 21:38
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] holly nuclei bat man

Happy New Year everyone!

 

Can we start a discussion once again on what causes holes in nuclei?  It is
pretty clear in my experience it has something to do with heating at least
for me with antigen retrieval.  When I do HIER at ph 8 especially I am
seeing a lot of holes in nuclei.  H&E on same tissue alone without HIER/IHC
doesn't seem to exhibit this artifact (holes in the nuclei).  I bet it can
also happen if the tissue is not fixed well enough to protect it from
paraffin processing but right now I am seeing it more after HIER, don't see
it as much on my IHC slides with no AR or when I use eier instead of hier.
Also notice that ph6 HIER and Ph9 HIER are not as bad as when I use Ph8 for
the same times and temps. 

 

Cheers,

 

Patsy

 

Patsy Ruegg, HT(ASCP)QIHC

Ruegg IHC Consulting, LLC

40864 Arkansas Ave

Bennett, CO 80102

Fax: 720-859-4110

 <mailto:pruegg <@t> ihctech.net> pruegg <@t> ihctech.net

 

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