Is my secondary antibody the cause for non-specific signal? - (Aug/21/2007 )

Hi all,

So I am trying to optimize my signal of endothelial cells stained for Flk-1 (a receptor on the membrane) by titrating both the secondary and primary antibodies. I set up my titration in a 96 well plate only to find that my negative control shows as much signal as most of the other concentrations. My primary antibody is brand new. In contrast, my secondary antibody expired a few months ago (I just wanted to try to see if I could still use it, probably a mistake in hindsight). I'm still new to immunofluorescence, so I was wondering if anyone knows if even though the secondary antibody probe seems to show signal, that it has gone bad due to the antibody losing specificity? I was just wondering if anything else could be the cause of non-specific binding prior to ordering more antibody (funding is tight). I fix with 3% formalin, rinse, block with 10% BSA prior to adding the primary antibody+ blocking solution, then rinse (3x5 min) then add secondary ab+ blocking solution, then rinse (3x5 min) and image.

Thanks,
TheDifferentiator

you could try a more potent block - in immuno histo/cryo we use animal serum specific to the secondary antibody (eg goat anti rabbit we use goat serum)

old age tends to be less binding full stop (as long as your washes are good)

dom

some secondary antibodies have more background. You need a better blocking agent.

Thank you for your responses. I'll see if a better blocking agent helps.