For immunohistochemistry, the eyes were evaluated at 5 hours, 7 and 90 days after low-energy laser treatment (0.065 mJ). Specifically, the eyes were enucleated and the anterior segment and vitreous were removed. The posterior eyecups (
n ≥ 5 per experimental group) were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB) for 30 minutes and cryoprotected in graded sucrose (10%, 20%, 30%) in PB overnight.
33 For retinal wholemounts, the entire eyecup (retina/choroid/sclera) was processed for immunohistochemistry as described previously
32,34 and specifically below. For retinal sections, the eyecup was embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura, Torrance, CA, USA), frozen at −20°C, and sectioned transversely at 14 μm on a Microm HM550 cryostat (Thermo Scientific, Walldorf, Germany). Retinal sections were collected onto polylysine-coated slides (Thermo Scientific, Soresby, VIC, Australia) and stored at −20°C. For labeling, frozen sections or whole mount eyecups were washed in PB and blocked with 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), and 0.5% Triton X-100 in 0.1 M PB (pH 7.4) for 1 hour. The samples were then incubated with primary antibodies or lectins (described below) diluted in 3% NGS, 1% BSA, and 0.5% Triton X-100 in PB overnight at room temperature (sections) or 4 days at 4 °C (whole mounts). Blood vessels were labeled by using the lectin
Bandeiraea simplicifolia BS-I Isolectin B4-FITC conjugate (IB4, 1:75, catalogue No. L2895; Sigma-Aldrich Corp., St. Louis, MO, USA). The RPE was labeled with Alexa Fluor 633-Phalloidin, a high-affinity F-actin probe that labels RPE cell membranes (1:200, Phalloidin, Cat. No. A22284; Life Technologies, Scoresby, VIC, Australia). Microglial cells were labeled by using an antibody against ionized calcium-binding adapter molecule 1 (1:1500, rabbit anti-IbA1, immunogen, synthetic peptide corresponding to the C-terminus of human IbA1: PTGPPAKKAISELP, Cat. No. 019-19741; Wako Pure Chemical Industries, Richmond, VA, USA). After labeling, and washing with PB, sections/whole mounts were incubated with secondary antibodies as required (1:500, goat anti-rabbit IgG Alexa 568; Life Technologies Australia, VIC, Australia) and DAPI nuclei stain (1:3000; Life Technologies Australia) for 1 hour (sections) or overnight (whole mounts). The samples were washed in PB then mounted with fluorescence mounting medium (Dako, North Sydney, NSW, Australia) and covered with a glass coverslip.