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I am staining tissue sections and I did a mistake, I was supposed to have mixed 3 primary antibodies but I stained only with one of them. After the 1h incubation I washed 10 min with PBS and then I did another hour incubation with the 2 antibodies I forgot to add before. Can that affect my staining? I am afraid I wanted to test different dilution of the first antibody I added alone, and then I added another hour incubation with 2 more antibodies.

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Generally for IHC/ICC staining separately with the antibodies is not a problem. Staining with separate primary antibody steps is often done when the antibodies are from incompatible species and so might cross-react with each-other giving non-specific staining.

However, the 10 min wash and 1 hour incubation with the other antibodies may wash off some of first antibody, leaving you with weaker signal than expected. This may affect how "bright" your stain appears after the secondary antibody step.

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Is this an ELISA? In that case, I suggest you doing this experiment again because now you are incubating for an extra hour. Incubation time is set at fixed range because increasing it may also increase the background noise of your assay.

Incubation times - sensitivity may be increased with a longer incubation time at room temperature. Be aware that the top of the curve may flatten out and become unusable, limiting the assay range. Additionally, background may increase

Resources: https://resources.rndsystems.com/pdfs/datasheets/edbapril02.pdf

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    $\begingroup$ This is not an ELISA it is immunohistochemistry or immunocytochemistry. $\endgroup$
    – bob1
    Jun 2, 2021 at 0:50

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