Quantitative investigation of photobleaching-based autofluorescence suppression in formalin-fixed paraffin-embedded human tissue

Immunofluorescence (IF) is a crucial technique in histopathology, enabling the visualization of multiple antibody distributions within single tissue specimens. However, autofluorescence (AF), which originates from endogenous molecules in formalin-fixed paraffin-embedded (FFPE) tissue, poses a persistent challenge by interfering with the fluorescence signal of interest in IF analysis. While photo-irradiative bleaching has emerged as a protocol to suppress the AF signal, there have been minimal quantitative investigations into this method across various experimental conditions. In this study, we investigated the efficacy of a bleaching-based method for reducing AF in FFPE human tissues. This research analyzed AF intensity as a function of exposure time, deparaffinization, emission range, and tissue types. Furthermore, by employing fluorescence lifetime imaging microscopy, we isolated the IF signal from AF to facilitate accurate quantification. This study provides valuable insights into AF reduction methodology to improve the reliability of IF-based histopathological analyses and advance our understanding of tissue biology and pathology.