Fwd: [Histonet] RE: AP problem

From:Melanie Black

>I have also had rod-like crystals in my Alk phos/NBT system. I have 
>shown them to the company rep, who has no idea what they might be. 
>With so much written about it here...I think DAKO should sit up and 
>take a look.

Melanie Black

>Thanks all for the cat scratch advice. I am trying it with HRP- AEC. 
>Will report result.
>>>>  "C.M. van der Loos"  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 
>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" 
>Date  Fri, 15 Oct 2004 15:45:05 -0400
>To  histonet@lists.utsouthwestern.edu, jkiernan@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.
>----- Original Message -----
>>From  "Richard Cartun" 
>Date  Fri, 15 Oct 2004 16:45:57 -0400
>To  ,, 
>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 W. Cartun, Ph.D.
>Director, Immunopathology & Histology
>Assistant Director, Anatomic Pathology
>Hartford Hospital
>80 Seymour Street
>Hartford, CT  06102
>Histonet mailing list

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