I wish to probe for two targets using a conjugated and non-conjugated primary antibody (raised in different species) for my IHC experiment.
Question is, when would I include the conjugated primary antibody in the procedure? My initial thoughts were to combine the conjugated antibody (AF647 conjugated) with the secondary antibody (AF488) for the non-conjugated antibody. Alternatively, incubate the conjugated antibody after the secondary antibody step. Does anyone have experience of this?
N.B. I realise I should probably optimise this, but samples/reagents are limited...
Any advice would be greatly appreciated!
Present stepwise procedure to intend to follow. Otherwise, in immunohistochemical staing of tissues, you must add either conjugated or nonconjugated antibody after BSA/FCS i.e. nonspecific blocking step.
Non conjugated antibody eventually followed by antispecies conjugated antibody will amplify the reaction.
You may two antibodies with different specificities simultaneously.
Better shall be to use two antibodies with different specificities but conjugated to different flourochromes. Say one FITC and second Texas red bound, and use uv microscopy to visualize antigens/ cells in the tissue sections.
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