[Histonet] immunohistochemistry help please

[Tarantelli, Rebecca Anne]

Gudrun, 

I tried it again this morning with a new bottle of xylene, and it is taking up some hematoxylin, but very patchy. These are all tissues from the same block, cut in sequential slides. Any other suggestions? 


Rebecca



-----Original Message-----
From: Gudrun Lang [mailto:gu.lang <@t> gmx.at] 
Sent: Friday, August 02, 2013 7:40 AM
To: Tarantelli, Rebecca Anne
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: AW: [Histonet] immunohistochemistry help please

Are all slides from the same tissue sample with the same pretreatment?
I can't believe, that you don't have any hematoxylin-staining, even with insufficient deparaffination. It would rather be patchy and uneven.
Overoxidation of the dye? Change of the binding-sites in the tissue?
Acid-residues masked by Aminogroups?
I guess there's something wrong with your buffer.
Gudrun


-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Tarantelli, Rebecca Anne
Gesendet: Donnerstag, 01. August 2013 21:20
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] immunohistochemistry help please

Hi all,

I am new to this forum, but a friend recommended I send my issue out to you for help. Four weeks ago I completed a new protocol for Immunohistochemistry
(IHC) using protinase K epitope retrieval, and monoclonal antibody 3F6. It worked perfectly. Three days later, after 13 beautiful stains, it just stopped working. The slides will not even take up the counter stain - Hematoxylin. I have exchanged my xylene, and tried again, and it still is not working. I am guessing since my counter stain isn't working, this is an issue in the deparaffinization stage, but I'm just not sure.

Please let me know if you have any suggestions!

Thank you
Rebecca

Rebecca Tarantelli

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