Protocols for Reducing Autofluorescence and Improving Fluorescent Nuclear Staining in Mouse Testis

Abstract

Previously, we had encountered significant autofluorescence (AF) in Leydig cells in the testis. Leydig autofluorescence often represents a barrier to effectively analyzing and depicting the expression of proteins by testicular Sertoli cells. Biological AF is an intrinsic property of cells and tissues, and intrinsic autofluorescence of some tissues can serve as a useful diagnostic indicator in certain disease situations. However, it is often an obstacle to immunofluorescence‐based analyses by masking and interfering with specific fluorescent signals. Hence, the need to reduce tissue AF is imperative for a successful visualization of co‐localizing proteins. Therefore, we sought to utilize the fat‐soluble diazo dye Sudan Black B (SBB) to reduce undesirable autofluorescence in our samples. Following treatment of the testis tissue sections with Sudan Black B for 10–15 min, the intrinsic fluorescence was significantly masked, allowing specific staining of Sertoli and Leydig cells with antibodies against SOX9 and DDX4. Additionally, we sought to use an alternative fluorescent nuclear stain, as 4′,6‐diamidino‐2‐phenylindole (DAPI) produces insufficient labeling in the nuclear of testis cells fixed with Bouin’s solution, which is used more commonly than formaldehyde solutions in testis research. As a result, we have developed protocols to significantly 1) treat tissue with SBB to reduce autofluorescence, and 2) improve fluorescent nuclear staining in mouse testis, without changing fixative solutions. These protocols can be used separately or together and can be altered to fit optimal experimental parameters. The method developed in this study will improve the visualization of multiple AF signals.