Saturation of tissue before staining and buffer of incubation

Christelle

I want to stain my cleared mouse brain, but I look the protocol described in the publication, there is no saturation of the aspecific site with serum from the 2nd antibody specie. Has anybody tried to stain samples including this step before the incubation with the first antibody? has anybody tried to incubate the antibodies in other solution that the sodium borate buffer?
Cheers
Christelle

Shashank20

Hi @ Christelle, I tried blocking of the unspecific binding site with TBST buffer with goat serum, and further on incorporated goat serum in the primary and secondary antibody solutions. I landed up in huge autofluorescence. Now I plan to reproduce the exact staining conditions as mentioned in the paper ? How about you ?