Why do we need controls for immunofluorescence?

There are several reasons for needing controls in immunofluorescence. One reason is to ensure that the observed staining is accurate and not caused by factors like autofluorescence or non-specific binding. It's essential to include controls such as samples with no primary antibody or isotype controls. These controls serve as standards, making data interpretation more reliable, particularly in cases where signal detection is absent. Additionally, controls help assess and quantify background fluorescence levels, providing a baseline for comparison and aiding in the accurate interpretation of signal intensity. In endogenous controls,  both primary and secondary antibodies are not included in the buffers, revealing the level of autofluorescence in the sample.