High levels of autofluorescence in tissue samples can entirely mask specific labellings with fluorophores and thus impair immunofluorescence histochemistry. In pancreatic tissue samples we observed autofluorescence as a common problem often mediated by the fixation and processing procedure.

Using epifluorescence microscopy, we analysed the intensity and spatial distribution of autofluorescence in formalin‐fixed, paraffin‐embedded human pancreatic tissues and developed an efficient quenching method to reduce the unwanted light emission. The optimized quenching protocol using Sudan black B reduced the unequally distributed tissue autofluorescence to a low and intensity‐equalized background level. Quantitative image analysis demonstrated autofluorescence suppression by 65–95%, depending on the selected fluorescence filter setups. The procedure did not affect specific immunofluorescence labelling or tissue integrity. As a clear result of Sudan black B treatment, a tremendous improvement of the signal‐to‐noise ratio was achieved, allowing a reliable detection and quantification of specific fluorescent labels. Other tissue treatment methods, such as cupric sulphate, toluidine blue and ultraviolet irradiation, or combinations of these with Sudan black B, were not as efficient.

The easy‐to‐perform Sudan black B technique improves dramatically qualitative and quantitative fluorescence analysis of critical pancreatic tissue sections and rescues even overfixed tissues for immunofluorescence application.