Reduction of Retinal Pigment Epithelium (RPE) Background Autofluorescence With Sudan Black Enhances Visualization of Fluorescently–Labeled Proteins in ex vivo RPE Flatmounts

Abstract

Purpose: : To describe a novel biochemical technique for reducing background autofluorescence, caused by lipofuscin–like pigments, to improve visualization of human RPE ex vivo flatmounts by immunofluorescence confocal micrsocopy.

Methods: : Procedures on human tissue were approved by the Institutional Review Board (IRB). Several human donor eyes were obtained 8–12 hours post mortem from the Georgia Eye bank and fixed in 4 % paraformaldehyde. Using a dissecting microsope, the entire RPE layer was isolated from each eye with the subjacent choroid attached in ex vivo RPE flatmounts. To quench the autofluorescence of the RPE, specimens were blocked with 0.3 % Sudan Black in 70% ethanol for 7–10 minutes followed by ten rapid rinses with Tris buffer. Specimens were incubated for one hour with a monoclonal antibody directed against the intercellular junction protein zonula occludens–1 (ZO–1) followed by fluorescently–labeled secondary antibody. Specimens were mounted flat on glass slides in Prolong Antifade mounting medium, and labeling of ZO–1 was assessed by confocal miscroscopy. In addition to the Sudan Black–treated specimens, matched control specimens were prepared by the same immunofluoresence protocol without the Sudan Black blocking buffer.

Results: : For the control specimens, which were not treated with Sudan Black, large clusters of fluorescent lipofuscin–like granules were observed by confocal miscroscopy thoughout the RPE . In fact, this autofluorescence completely obscured visualization of ZO–1 in intercellular junctions of the RPE. In contrast to controls, Sudan Black–treated specimens demonstrated minimal background autofluorescence, and ZO–1 was clearly observed at intercellular junctions encircling the RPE cells.

Conclusions: : Sudan Black significantly reduces background autofluorescence in human ex vivo RPE flatmounts thereby enhancing visualization of fluorescently–labelled proteins by confocal micoscopy.

Keywords: retinal pigment epithelium • cell adhesions/cell junctions • microscopy: light/fluorescence/immunohistochemistry