What to do with high autofluorescence background in pancreatic tissues - an efficient Sudan black B quenching method for specific immunofluorescence labelling

Histopathology
Till ErbenJürgen Schnekenburger

Abstract

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...Continue Reading

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Citations

Nov 13, 2018·The Journal of Reproduction and Development·Manami SenooMasahito Ikawa
Oct 9, 2018·Archives of Pathology & Laboratory Medicine·Wenxia SuJinqin Liu
Oct 24, 2017·Current Protocols in Neuroscience·Niteace C Whittington, Susan Wray
Mar 18, 2020·Expert Review of Molecular Diagnostics·Vesal YaghoobiMalini Harigopal
Aug 30, 2019·Histochemistry and Cell Biology·Matthew J MoldChristopher Exley
Sep 13, 2020·Journal of Neuroscience Methods·Nicholas F NoltaMartin Han

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