[Histonet] RE: AP problem

[Joanne Mauger]

Thanks all for the cat scratch advice. I am trying it with HRP- AEC. Will report result. 
Jo


>>> "C.M. van der Loos" <c.m.vanderloos <@t> amc.uva.nl> 10/17/04 02:20PM >>>
Dear Joanne,

Usually, AP staining doesn't show up in a crystal-like precipitate, but I have seen it a couple of times. Never found out what was the cause of it. Be sure that after your IHC procedure and before the AP visualization, the sections are washed at with a Tris-HCl pH8.5 buffer. Phosphate ions from PBS will inhibit your AP reaction. You also may mix the Permanent Red components first and than press immediately through a syringe-filter to remove all bigger particles in the AP solution.
Have you viewed your slides under a fluorescence microscope? A small amount of Permanent Red reaction product also gives a bright red fluorescence signal when using the rhodamine filter pack (green light). 
Good suggestion by Richard to use a tonsil as a negative control. But, how about running the good old control blanc slide without any primary antibody?
Hope this helps.

Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center
Amsterdam - The Netherlands    

----- Original Message ----- 
>From  "Joanne Mauger" <MAUGER <@t> email.chop.edu> 
Date  Fri, 15 Oct 2004 15:45:05 -0400 
To  histonet <@t> lists.utsouthwestern.edu, jkiernan <@t> uwo.ca 
Subject  Re: [Histonet] IHC withquantum dots 
Hi group,
Can anyone help me with suggestions-I am working up the monoclonal Ab (H2A10) to cat scratch disease. Ab from Novus Biologicus. I have a purchased control from Newcomer Supply. I am using permanent red chromagen from Dako., with alk phos link also from Dako. I am getting a nice specific staining of what looks like specs vs tiny rods or spirochetes, but the control tissue does , by Warthin Starry, look much more spirochete-ish. Can I trust this result? I have also stained several suspected + cases and see the same dust like + staining,but I am afraid it is teeny bits of precipitate. 
Does this ring a bell?
Please help.
Thanks,
Jo

----- Original Message ----- 
>From  "Richard Cartun" <Rcartun <@t> harthosp.org> 
Date  Fri, 15 Oct 2004 16:45:57 -0400 
To  <MAUGER <@t> email.chop.edu>,<histonet <@t> lists.utsouthwestern.edu>, <jkiernan <@t> uwo.ca> 
Subject  Re: [Histonet] IHC withquantum dots 
Have you tried staining a section of tonsil to see if you get the same pattern of staining.  I have found "precipitation" to be a problem with alkaline phosphatase (PA) detection, although I am by no means an expert when it comes to AP IHC.  I use HRP/AEC for most of my infectious disease studies including Bartonella henselae.  Don't forget that you may not see the normal morphology of the bacterium with IHC.  You are labeling epitopes on the surface of the organism; you are not staining the entire cell wall.  I have found B. henselae to be one of the most difficult organisms to label (and interpret) with IHC.  Good luck!

Richard 


Richard W. Cartun, Ph.D.
Director, Immunopathology & Histology
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102