One realisation is that my protocol with 10%NGS in PBST is not working as well as the IC solution. I need to prepare that solution independently but I do not have the recipe. My protocol is fix-permeabilize-block-primary Ab in blocking overnight incubation- secondary antibody incubation- HOECHST and imaging. Washes are done in 1x PBS. In IC protocol, fix-2.5%NGS inactivated blocking in IC soln -primary ab in IC solution o/n incubation-secondary ab in IC soln- HOECHST in PBS- imaging. In IC protocol, washes are done in IC solution.

Both protocols were performed simultaneously on the same type of cells under the same conditions including the dilutions.