Retinal Vascular Amyloidosis Immunofluorescence

Immunofluorescence staining was performed on transverse retinal sections, as previously described [23 (link)]. Briefly, the slides were deparaffinized, boiled for 20 min in a citrate antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), and blocked with 10% normal donkey serum. According to previous studies investigating retinal vascular amyloidosis, CD31 was used as a marker for vascular endothelium [24 (link),25 (link)]. The retinal tissue was incubated with the primary antibodies: rat anti-CD31 (1:100; Abcam ab56299, RRID: AB_940884) and rabbit anti-Aβ42 (1:100; Cell Signaling Technology Cat# 14974, RRID: AB_2798671), and incubated with a corresponding fluorescence-conjugated secondary antibody and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (nuclear marker, color blue) (Jackson ImmunoResearch, West Grove, PA, USA). No staining was observed in the imaging of the negative control: staining performed without primary antibody. The sections were visualized with a confocal microscope (LSM880, ZEISS Microscopy, Jena, Germany) using 63× magnification.