Characterizing and Quenching Autofluorescence in Fixed Mouse Adrenal Cortex Tissue.

Tissue autofluorescence of fixed tissue sections is a major concern of fluorescence microscopy. The adrenal cortex emits intense intrinsic fluorescence that interferes with signals from fluorescent labels, resulting in poor-quality images and complicating data analysis. We used confocal scanning laser microscopy imaging and lambda scanning to characterize the mouse adrenal cortex autofluorescence. We evaluated the efficacy of tissue treatment methods in reducing the intensity of the observed autofluorescence, such as trypan blue, copper sulfate, ammonia/ethanol, Sudan Black B, TrueVIEW Autofluorescence Quenching Kit, MaxBlock Autofluorescence Reducing Reagent Kit, and TrueBlack Lipofuscin Autofluorescence Quencher. Quantitative analysis demonstrated autofluorescence reduction by 12-95%, depending on the tissue treatment method and excitation wavelength. TrueBlack Lipofuscin Autofluorescence Quencher and MaxBlock Autofluorescence Reducing Reagent Kit were the most effective treatments, reducing the autofluorescence intensity by 89-93% and 90-95%, respectively. The treatment with TrueBlack Lipofuscin Autofluorescence Quencher preserved the specific fluorescence signals and tissue integrity, allowing reliable detection of fluorescent labels in the adrenal cortex tissue. This study demonstrates a feasible, easy-to-perform, and cost-effective method to quench tissue autofluorescence and improve the signal-to-noise ratio in adrenal tissue sections for fluorescence microscopy.