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Improving primary Ab specificity?

Improving primary Ab specificity? - Immunohistochemistry Forum

Improving primary Ab specificity? - Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections.


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Old 11-27-2008, 12:06 PM
Pipette Filler
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Default Improving primary Ab specificity?



Hi all
Can anyone give me any information about increasing the specificity of my primary antibody? I'm doing DAB staining of human paraffin sections; after 20 minutes block and 2 hrs at room temperature (1 in 100) with the primary I get diffuse staining across all of the sample. The no primary negative control doesn't have any stain. Any ideas for changing conditions/buffers etc much appreciated as I've never done any IHC before! Thanks
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Old 12-03-2008, 11:44 PM
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Default Re: Improving primary Ab specificity?

It seems that you have background staining?, maybe a exces of antibody?, which is the antibody (reference and company)? (do you observe the expected staining pattern, nuclear,membrane...), and what is your tissue.
If you are unmasking the antibody, with the microwave sometimes you can obtain background staining.
You can reduce background staining with overnigth incubation (4ēC) and higher antibody dilution.
I recomend you a complete antibody titration (five or six dilutions) and at least two unmasking buffers to begin (citrate buffer ph 6, EDTA ph 8).After unmasking in the microwave always cool the solution slow before beginning with the IHC (20-30 minutes).
Always run positive and negative controls (two tissues in the same slide better than one) in each assay.
For signal amplification you can try Envision (Dako) or another more sensitive substrate like nickel-Dab for HRP of NBT for PA.
The negative control is ok so you don´t have problems with endogenous peroxidase,biotin or crossreactivity of the secondary antibody, the problem must be the primary antibody.
I hope this could help you and sorry for my english...
Best Regards
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Old 12-04-2008, 02:25 PM
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Default Re: Improving primary Ab specificity?

thanks very much - I'll give your suggestions a try. the antibody is Imgenex OPG, it's a secreted protein, and they're skin sections. I've tried microwave antigen retrieval but it damaged my sections too much, I repeated without a retrieval step but am considering trypsin digestion. Hopefully overnight at a higher dilution will help. Thanks again and best wishes!
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Old 12-04-2008, 06:36 PM
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Default Re: Improving primary Ab specificity?

We are working with skin sections now and we have no problems with the antigen retrieval , we always use 10% poly-l-lysine or vectabound treated slides for IHC, it prevents tissue loss during the unmasking procedure and preserves the morfology. You could even try with both adhesives in the same slide (vectabound plus pure poly-L-lysine) if you still have problems. Instead of 20 min at 600W you can also try 5 min at 800W for example.
Our problem with the skin (mouse) is the Keratin unspecific staining with some antibodies, we are working on it...
Best regards and good luck!
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