The retina was sectioned and IHC was performed as described previously.23 (link) GA was assessed by masked observers using the scoring system based on methods reported previously by Anderson et al.24 (link) A GA score of 4 or 5 indicated a high degree of GFAP, whereas a score of 1 to 3 indicated a low degree of GFAP staining. Apoptosis was evaluated by TUNEL. The number of TUNEL-positive cells (green) was recorded in three fields of each retina that covered 212 × 212 μm. Images were acquired with a confocal laser scanning microscope (FV1000, Olympus. Hamburg, Germany).
Microglial activation was assessed by ionized calcium-binding adapter molecule 1 (Iba-1) immunofluorescence. Retina sections were incubated overnight in a humid chamber at 4°C with goat anti-Iba-1 (Abcam, Madrid, Spain). Then, samples were washed and incubated for 60 minutes with anti-goat Alexa 488 (1:200; Molecular Probes, Eugene, OR, USA). After washing, sections were mounted in Vectashield (Vector Labs, Burlingame, CA, USA) mounting medium with 4′,6-diamidino-2-phenylendole (DAPI). Images were acquired with a confocal laser scanning microscope (FV1000; Olympus) with a ×60 objective. Images size were 1024 × 1024 pixels. Fluorescent values were obtained with the software Olimpus Fluoview (v.4.2) in the confocal images (oib) with an ROI plugin and with background substraction.
Microglial activation was assessed by ionized calcium-binding adapter molecule 1 (Iba-1) immunofluorescence. Retina sections were incubated overnight in a humid chamber at 4°C with goat anti-Iba-1 (Abcam, Madrid, Spain). Then, samples were washed and incubated for 60 minutes with anti-goat Alexa 488 (1:200; Molecular Probes, Eugene, OR, USA). After washing, sections were mounted in Vectashield (Vector Labs, Burlingame, CA, USA) mounting medium with 4′,6-diamidino-2-phenylendole (DAPI). Images were acquired with a confocal laser scanning microscope (FV1000; Olympus) with a ×60 objective. Images size were 1024 × 1024 pixels. Fluorescent values were obtained with the software Olimpus Fluoview (v.4.2) in the confocal images (oib) with an ROI plugin and with background substraction.
