Hello everyone. I am having some problems with an immunofluorescence experiment. My aim is to use the IF-CBA method for human samples and get negative or positive results.

Please check these reserch to learn about some details about IF-CBA（DOI:10.3389/fneur.2023.1289810 or DOI:10.21037/atm-21-3072；these two can read on resarchgate.）

My experimental system uses HEK293T with PCdna3.1 plasmid transfected with characteristic proteins (e.g. AQP4), then fixed (4% paraformaldehyde), washed and incubated with Triton 100x (0.3% Triton-100, 2h) and last closed (5%BSA, 1h). Subsequently, different experiments were performed. When I stained with the standardized antibody and the corresponding fluorescent antibody（both from abcam）, it showed good results; however, when I next incubated with human serum as the primary antibody, there was no significant fluorescence from either the FITC-labeled human IgG antibody or the cy3-labeled human IgG antibody. Why is this phenomenon occurring when the human original sample is known as a certain positive sample? Should I perform antigen repair? Thanks for reading.