Renal tissue emits intense autofluorescence, making it difficult to differentiate specific immunofluorescence signals and thus limiting its application to clinical biopsy material.
To identify and minimize autofluorescence of renal tissue and demonstrate a simple, efficient method to reduce autofluorescence using Sudan black B.
In this study, the sources and features of autofluorescence emitted from kidney tissue were examined. Broad autofluorescence was visualized in both frozen and paraffin kidney sections of normal mice and mice with Adriamycin-induced nephropathy using confocal laser scanning microscopy. Autofluorescence appeared in commonly used 4',6-diamidino-2-phenylindole, fluorescein isothiocyanate, and Texas Red channels but not in far-red channel, and emitted extensively from red cells, injured tubulointersitial cells, and protein casts in diseased kidney. To eliminate autofluorescence, Sudan black B was used on formaldehyde-fixed paraffin sections and frozen sections of mouse kidney. The effects of Sudan black B in various concentrations were tested on kidney tissue.
The 0.1% Sudan black B effectively blocked autofluorescence from both paraffin and frozen sections without adversely affecting specific fluorescence signals. Interestingly, the solvent for Sudan black B, 70% ethanol, was also shown to reduce autofluorescence on frozen sections, but not on paraffin sections.
This study demonstrates a simple, efficient, and cost-effective method to reduce autofluorescence using Sudan black B, and also provides a comprehensive approach to identify and minimize autofluorescence of renal tissue.