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. <\/p>\n
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,<\/p>\n
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<\/p>\n
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. <\/p>\n
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 <\/p>\n
*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. <\/p>\n
I've also checked the filters and those all are correct for the anitbodies that I'm using. <\/p>\n
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More here: 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. Continue reading
\nBiochemistry - reddit<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"