Iba 1

Histological sections of the mouse retina were analyzed after specific immunofluorescence labeling with a monoclonal antibody to TNF-α (1:500; Abcam), as previously described.^{19 (link),22 (link)–24 (link, link)} In addition, specific antibodies against glial fibrillary acidic protein (GFAP) or Iba1 (1:500; Santa Cruz, CA, USA) were used to identify astroglia and microglia, respectively. A mixture of Alexa Fluor 488- or 568-conjugated IgGs (1:500; Thermo Fisher Scientific, Waltham, MA, USA) was used for the secondary antibody incubation. The 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Thermo Fisher Scientific) was used for nuclear counterstaining. Slides were examined by fluorescence microscopy and images were recorded by digital photomicrography (Carl Zeiss, Thornwood, NY, USA). Negative controls were performed by replacing the primary antibody with serum or using an inappropriate secondary antibody to determine species specificity.

The cortex was fixed overnight using 4% paraformaldehyde at 4˚C and dehydrated in 30% sucrose at 4˚C for 2-3 days. After embedding in optimal cutting temperature compound, 7-µm coronal sections were cut within 1-3 mm posterior to the bregma and placed on gelatin-coated microscope slides. All sections were frozen at -20˚C before use.
For immunofluorescence analysis, all sections were blocked using 10% serum-blocking buffer (0.1% Triton X-100, 3% (w/v) bovine serum albumin (BSA) and 0.05% Tween-20) for 2 h at room temperature. Then, the sections were incubated with primary antibodies against AATF (1:100; cat. no. WH0026574M4; Sigma-Aldrich; Merck KGaA), NeuN (1:100; cat. no. SAB4300883; Sigma-Aldrich; Merck KGaA), glial fibrillary acidic protein (GFAP; 1:100; cat. no. sc-33673; Santa Cruz Biotechnology, Inc.), ionized Ca²⁺-binding adaptor molecule 1 (Iba-1; 1:100; cat. no. sc-32725; Santa Cruz Biotechnology, Inc.) and cleaved-caspase-3 (1:100; cat. no. sc-3073; Santa Cruz Biotechnology, Inc.) overnight at 4˚C. The sections were next incubated with FITC- and Tetramethylrhodamine-conjugated secondary antibodies (1:1,000; cat. no. F0257; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature. The sections were dehydrated and covered with coverslips, and the slides were photographed under a 20x objective using a Leica DM4000B fluorescence microscope (Leica Microsystems GmbH).