4.2. Immunofluorescence

B1R, B2R, iNOS, fibrosis markers (α-smooth muscle actin (α-SMA) and collagen1 α) and glial cells markers (glial fibrillary acidic protein (GFAP) for macroglia and ionized calcium-binding adapter molecule 1 (Iba1) for microglia) were detected by immunohistochemistry on paraffin embedded retinae, as described previously [17]. Sections obtained from the Eye Biobank were deparaffinized prior to the immunofluorescence procedure. Glass slides were first incubated in sodium citrate buffer at 95 °C for 35 min. Sections were then left to cool down for 20 min at room temperature (RT), then washed three times (3 × 5 min) with 0.1 M PBS buffer (pH 7.4) and incubated for 1 h at RT in blocking buffer (PBS containing 10% donkey serum or goat serum and 0.25% triton X-100). Sections were left incubated overnight at RT, with the blocking buffer containing primary antibodies (Table 2).

List of primary antibodies.

The following day, slides were washed 3 × 5 min in PBS 0.1M and then incubated for 2 h at RT with secondary antibodies: Alexa Fluor 555 goat anti-rabbit (1:200, A21428), Alexa Fluor 488 goat anti-rabbit (1:200, A21206), Alexa Fluor 555 donkey anti-mouse (1:200, {"type":"entrez-protein","attrs":{"text":"A31570","term_id":"85652","term_text":"pir||A31570"}}A31570), Alexa Fluor 488 donkey anti-mouse (1:200, A21202), Alexa Fluor 546 donkey anti-goat (1:200, A11056), Alexa Fluor 555 goat anti-chicken (1:200, A21437), Alexa Fluor 633 donkey anti-goat (1:200, A21082), all purchased from Life Technologies, Burlington, ON, Canada. To eliminate the autofluorescence caused by tissue components and by the accumulation of lipofuscin [68], an autofluorescence eliminator reagent (2160, Sigma Aldrich, Oakville, ON, Canada) was used. The slides were then washed and mounted using a homemade glycerol solution. Images were obtained with a confocal microscope Zeiss-LSM800 equipped with an argon laser (Carl Zeiss, Jena, Germany) and transferred to a computer and analyzed using NIH ImageJ 1.36b Software (NIH, Bethesda, MD, USA). Images were obtained at 40× and 60× objectives. Semi-quantification of immunofluorescence staining intensity was made on five randomly selected surface areas of each retina from five dry AMD, five wet AMD and five controls. Background intensity (gray intensity) was subtracted from each individual value.