Histochoice

Five 5-μm FFPE sections were pooled together and placed in a 50-ml tube. The tissue sections were dedeparaffinized with HistoChoice (no. H2779, Sigma-Aldrich) three times, 10 min each. Samples were then hydrated gradually an alcohol series: wash in 100% ethanol (twice, 5 min each), 95% ethanol (once, 5 min), and 70% ethanol (once, 5 min). Samples were then washed in deionized water (three times, 5 min each). For antigen retrieval, samples were placed in 10 ml of tris-EDTA 0.05% Tween buffer (10 mM, pH 9) and heated in a boiling water bath (95°C) for 30 min. Samples were then washed in deionized water (three times, 5 min each).
FFPE samples were then suspended in 1 ml of 1× PBS and seeded in a flat 96-well plate at 50 μl of sample per well. The culture plate was then dried in an oven (60°C for 1 hour). Surface area coverage was determined with the JuLI Stage Recorder and analyzed with JuLI Stat software using the growth curve function to analyze the coverage (square millimeter per well) of samples in each well. Sample wells were then blocked overnight with 2% bovine serum albumin in 1× PBS. For coculture, 100,000 (at density of 1 × 10⁶ cell/ml) IcAR-PD1 cells were seeded on top of the FFPE samples for 24 hours. Supernatants were collected 24 hours later, followed by testing with a murine IL-2 commercial sandwich ELISA kit (BioLegend).

ESC colonies were disaggregated by incubation in 0.05% trypsin/EDTA for 3–5 min, washed in PBS, pelleted, and frozen at −80°C. Genomic DNA was isolated by stirring ESCs in 100 μL of 10% Chelex 100 solution in deionized water at 98°C for 15 min, as described earlier. Reactions were carried out as described previously for genotyping of animals. Karyotyping required ESC colonies to be incubated in a medium that contained 10 mg/mL colchicine (Sigma–Aldrich) at 37°C for 1.5 h. Next, ESCs were disaggregated in 0.05% trypsin/EDTA for 3–5 min, washed in PBS, suspended and incubated in 1 mL 0.56% KCl (Sigma–Aldrich) at room temperature for 20 min, and then fixed with methanol:acetic acid solution (3:1) at 4°C for 16 h. Finally, ESCs were dropped onto warm slides and stained with the Giemsa reagent (Merck) according to the manufacturer's protocol. Next, specimens were dehydrated in HistoChoice (Sigma–Aldrich), mounted with the VectaMount Mounting Medium (Vector Laboratories), and analyzed using transmitted light microscopy (Axioskop; Zeiss). For each ESC line, at least 30 metaphase plates were analyzed.

The eye balls were enucleated after sacrifice and frozen at −80°C and cryosectioned (Leica Cryostat, Buffalo Grove, IL, USA). Immunohistochemistry was performed to analyze the protein expression changes in the retinal layers viz. rhodopsin (Santa Cruz Biotechnology, Dallas, TX, USA), Thy1 (Santa Cruz Biotechnology), brain-derived neurotrophic factor (BDNF; Santa Cruz Biotechnology), and ciliary neurotrophic factor (CNTF; Santa Cruz Biotechnology). The sections were fixed using HistoChoice (Sigma-Aldrich) and then incubated with primary antibody (1:100 dilution) at 4°C overnight. Next day, the sections were kept in Cy3 labeled secondary antibody solution (1:200 dilution) (Jackson Immunoresearch, West Grove, PA, USA) for half an hour and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) (1:1,000 dilution) (Table 1). The sections were subsequently imaged under a confocal microscope to analyze the protein expression.