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. Brittani Heater is a Surgical Coordinator, Insurance Specialist, Patient Care Supervisor at Mandell Retina Center based in Elizabeth City, North Ca rolina. In fact, this autofluorescence completely obscured visualization of ZO–1 in intercellular junctions of the RPE. Mandell Retina Center Mandell Retina Center 110.09 miles (757) 227-4300. Surgical Coordinator, Insurance Specialist, Patient Care Supervisor at Mandell Retina Center. In addition to the Sudan Black–treated specimens, matched control specimens were prepared by the same immunofluoresence protocol without the Sudan Black blocking buffer.įor 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. Specimens were mounted flat on glass slides in Prolong Antifade mounting medium, and labeling of ZO–1 was assessed by confocal miscroscopy. 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. 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. Using a dissecting microsope, the entire RPE layer was isolated from each eye with the subjacent choroid attached in ex vivo RPE flatmounts.
Several human donor eyes were obtained 8–12 hours post mortem from the Georgia Eye bank and fixed in 4 % paraformaldehyde. Procedures on human tissue were approved by the Institutional Review Board (IRB). 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. 1 Albemarle Eye Center - Elizabeth City 1503 N ROAD ST, Elizabeth City, NC 27909 (757) 727-4300 Mandell Retina Center PC, Virginia Beach VA.
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