Ep373y

Immunohistochemistry experiments were performed on frozen human tissue sections fixed in 4% paraformaldehyde [35 (link)]. Infant (n=3) and aged adult (n=5) maculas were evaluated. Sections were blocked with 1 mg/mL of bovine serum albumin for fifteen minutes and were subsequently incubated with 50 μL of 1X TrueBlack autofluorescence quencher (Biotinium, Hayward CA) for 60 seconds followed by three five-minute washes, according to the manufacturer’s instructions. This treatment quenches the very abundant autofluorescence present in adult RPE cells. Sections were incubated with anti-CD34 (1:100, Abcam, EP373Y) and anti-ICAM1 (1:100, Developmental Studies Hybridoma Bank, Iowa City, IA, P2A4) primary antibodies for one hour. Negative controls were additionally obtained by omitting each primary antibody. After washing, Alexa-546-conjugated anti-mouse IgG (1:200, Invitrogen) and Alexa-488-conjugated anti-rabbit IgG (1:200, Invitrogen) secondary antibodies resuspended in PBS supplemented with 100 μg/mL diamidino-phenyl-indole (DAPI, Sigma) were added to each section for 30 minutes. Sections were washed and coverslipped, and photographs were acquired with a confocal microscope (Leica DM 2500 SPE). In all steps, from immunofluorescent labeling to finalization of figures, all infant and adult samples were treated identically.