I've been troubleshooting this protocol for awhile and I'm getting a good signal for Hoechst, but everything else either doesn't show a very good signal or has non-specific binding. Moreover, when I'm imaging it looks like every cell is fluorescent in every filter.

I'm trying to stain for myodifferentiation (MF20), apoptosis (CC3), and cell proliferation (Ki67). If someone has any suggestions please let me know! My protocol so far has been the following,

I've tried 2 different blocking/permeabilization buffers:a) 1% fish skin gelaton in PBS-T; b) 8% BSA + 10% horse serum + 0.25% Triton-X 100 in PBS

The 1st row I have Hoechst only, the 2nd row has Ki67 + Hoechst, the 3rd row has MF20 + Hoechst, the 4th row has CC3 + Hoechst, the 4th row has CC3 + Hoechst + drug, the 5th row has Hoechst + Ki67 + MF20 + CC3, the 6th row has Hoechst + Ki67 + MF20 + CC3 + drug. I do serial dilutions for each row- 1:100, 1:400, 1:600.

Antibodies that I'm using:Myosin 4 Monoclonal Antibody (MF20) Alexa Fluor 488;Cleaved Caspase-3 Alexa Fluor 647 Rabbit;Ki-67 Monoclonal Antibody (SolA15) eFluor 570, eBioscience;Hoechst 33342

*for MF20 I do a separate plate where I seed the cells in growth media, change the media to differentiation media (DMEM/F12 + 2% horse serum + 1% anti-anti) the next day, and allow cells to differentiate for 3-7 days then fix, permeabilize, block, and stain the cells.

I've also checked the filters and those all are correct for the anitbodies that I'm using.

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Biochemistry - reddit