I never dry my samples, but if I forget about DAPI before coversliping, I simply add some DAPI in 50% glycerol + PBS solution I usually put onto sections to prevent drying and then coverslip. For me, your plan sounds pretty good. But you can possibly lose some ABs complexes which will weaken your signal.

Snezhanna Medvedeva

Hi Snezhanna. I appreciate your reply so much!

I forgot to mention that I took my sections out of 2ary antibody, and put them immediately in a petri dish of 0.3% triton X which is what we use to mount free floating sections.

I'm concerned what the Triton X may have done to my samples, which would have contained excess 2ary (since I did not rinse with KPBS before mounting). My logic is that, perhaps the Triton X further permeabilized the tissue, and since the tissue was covered in excess 2ary, the signal would be crazy.

However.. the 2ary would primarily only bind to the 1ary antibody (especially since a blocking step with NDS was already done).

Any thoughts on this?

Either way, I'm going to try several things for the slices and see what happens!

Laura Loera-Lopez

I'm sure that it's okay, if your target protein for pABs is not located on the surface of membranes. I permeabilize my samples with Triton X100 at a blocking stage for 2-3 hours and then add pABs for a night in PBS containing 0,3% Triton + 0,5% BSA. So my samples contact with Triton overnight and nothing bad happens. :) Likewise with sABs.