I am detecting β-galactosidase in FFPE mouse heart tissue using a rabbit anti-β-gal primary antibody and a goat anti-rabbit Alexa Fluor 594-conjugated secondary antibody, imaged on a confocal microscope with the Rhodamine filter set. I am struggling to optimize acquisition parameters (laser power, detector gain, pinhole size, and z-stack settings) to obtain a clear, reproducible signal with minimal background.

At this stage, I also find it difficult to distinguish which parts of the fluorescence are true β-galactosidase signals and which may be artifacts or nonspecific background?

From a publication standpoint, I would like to ask whether reviewers in cardiovascular studies typically expect detailed localization and co-localization analyses, or if robust β-galactosidase signal detection and quantification alone (using 20× and 40× objectives) would generally be considered sufficient.?