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Author Topic: Problem in concentration of antibody  (Read 3449 times)

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Problem in concentration of antibody
« on: February 20, 2011, 03:49:56 AM »
   
I have done the optimization of concentration of CD45 for infarcted cardiomyocyte of rat. The diluents of the antibody were 1:20, 1:50, 1:80, 1:100, 1:150 and 1:300.

The antigen retrieval, w/ 0.1M sodium citrate buffer in mircowave oven for 2 mins high power and 10 mins low power, has used. The polycloned 2nd antibody and DAB have used. Also the positive control have stainned.

However the appearance of the negative control and the 1:20 (the most concentrate) are similar. Many brown spots and black spots appear in the cells on both slides which look like non-specific staining. Why?

Problem in concentration of antibody
« on: February 20, 2011, 03:49:56 AM »

Offline gula

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Re: Problem in concentration of antibody
« Reply #1 on: February 20, 2011, 05:23:56 AM »
Have you done any procedure to block endogen peroxidase acitivity or - in case you use a biotin-streptavidin detection system - to block endogen biotin binding?

If not, look at the protocols-part of IHC-World.
gula

Re: Problem in concentration of antibody
« Reply #1 on: February 20, 2011, 05:23:56 AM »