I am doing IHC on whole mount retina with a secondary antibody conjugated to AlexaFluor 488. I had a weak signal and I wonder if I could wash my retina in PBS-Triton or in a stripping buffer to re-incubate the retina with the same secondary antibody for a longer time to increase the signal.
Depending of how I wash my retina, I wonder 1) would I remove the secondary antibody from the first incubation ? 2) would I remove the primary antibody ? 3) Should I re-do the whole incubation process (primary + secondary antibody + hoechst). 4) would I damage my retina ?
Thanks for your help.
It seems likely that for your purpose, you may just need to add a (freshly diluted) secondary antibody for further incubation without removing an existing secondary antibody (c.f., such action is needed for re-probing with different primary or secondary antibody), but I am wondering whether this helps increase signals (green here). Given your appropriate selection of the secondary antibody (e.g., for species cross-reactivity), an increase in concentration (less dilution) of primary and/or secondary antibody, an incubation time, and an incubation temperature (e.g., from 4˚C to room temperature, or from room temperature to 37˚C) may generally lead to an increase in signals, albeit at the cost of an increase in the background signals (so, the suitable blocking condition is also needed).
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