[Histonet] IgA, IgM, IgG

[Jan Shivers]

MaryAnn,

I use tonsil control for dog IgG and IgM; I use stomach control (mucosa) for 
dog IgA.

Your dilutions of Abs will, of course, depend upon what the original 
concentration is of your stock Ab and vendor prep.  However, not all 
companies list the stock immunoglobulin concentration of their antibodies, 
so you'll just have to work them up and optimize dilutions for best staining 
results.  Here's what I use currently, but these change with change of 
vendor and change of Ab lot number:

Dog IgG - 1:6000; 0.4ug/ml working concentration
Dog IgM - 1:4000; (no stock Ig concentration given by manuf.)
Dog IgG - 1:1200; (no stock Ig concentration given by manuf.)

I use Proteinase K (Dako) enzyme digestion for 5', room temp, to unmask 
antigens for all 3 antibodies.

Yes, you'll get background with these specific antibodies, but you can 
decrease it by adding 4% normal dog serum in your 'linking or secondary' Ab 
(per volume).

Hope this helps.

Jan Shivers
Section Head - IHC/Histo/EM
Veterinary Diagnostic Laboratory
College of Veterinary Medicine
University of Minnesota
St. Paul, MN
shive003 <@t> umn.edu



----- Original Message ----- 
From: "MaryAnn Dixon" <DixonM <@t> vetmed.ufl.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, November 20, 2007 1:16 PM
Subject: [Histonet] IgA, IgM, IgG


I am working up anti dog IgA, IgG and IgM antibodies and was wondering what 
the better control tissues would be for these.  I have been using tonsil for 
all three but would love some input on any that work better.  Also, what is 
everyone using for pretreatment?  I'm teetering between prot. K and no 
treatment at all. The dilutions of these antibodies are getting higher too. 
My IgG was out to 1:6000. Do these antibodies generally have some accepted 
background with them?  I thought I was told that they do and you cant get it 
out.  Can anyone please share some dilutions and pretreatments for these 3 
antibodies.  Don't care if they are for anti-human, just would like a 
starting point. I am using HRP mach 4 detection from biocare.


MaryAnn Dixon
Biological Scientist
Anatomic Pathology
University of Florida
School of Veterinary Medicine
352-392-2235 ext 4517

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