Immunofluorescence microscopy

After isolating on the first day, a small part of mixed retinal cells was seeded at a density of 1 × 10⁶ cells per well in a 24-well plate with poly-L-lysine-coated glass coverslips at the bottom. For cultured retinal microglia, we cultured them after shaking on day 17 at a density of 2 × 10⁵ cells per well onto poly-L-lysine-coated glass coverslips in a 24-well plate. After attachment to the coverslips, cells were deprived of medium and fixed with 4% PFA for 1 h. Then, 0.1% Triton was used for permeabilization for 20 min. Then, 5% bovine serum albumin (BSA, Sigma) was performed as a blocking buffer. Primary antibodies of anti-ionized calcium-binding adaptor molecule 1 (Iba1, Abcam, #ab178846, 1:200) and anti-Purinergic Receptor P2Y12 (P2RY12, Biolegend, #848001, 1:100) were used to stain the microglia-related marker at 4°C overnight. Primary antibodies were incubated at 4°C overnight. The next day, coverslips or tissue sections were incubated with secondary antibodies (Alexa Fluor 488 of goat anti-mouse, Yeasen, 1:1,000; Alexa Fluor 647 of goat anti-rabbit, Yeasen, 1:1,000) at room temperature for 1 h. DAPI (Abcam, #ab228549, 1:1,000) was used for staining nuclear DNA. The morphology of retinal microglia and their specific locations in retina layers were observed under immunofluorescence microscopy (Leica) equipped with the NIS-Elements Advanced Research software.

Mixtures of ChipEXO at 2-fold serial dilutions and hCoV-19/Turkey/ERAGEM-001/2020 strain of SARS-CoV-2 at a fixed dose of 100 FFU were incubated at 37°C for 1 h. The mixtures were then added in triplicate to confluent Vero E6 cell monolayers. After absorption for 1 h at 37°C, the supernatants were removed and the cells were washed with PBS. The cell monolayers were overlaid with a medium containing 1% CMC and then incubated at 37°C with 5% CO₂ for 24 h. The remaining steps of FFA were performed as described above for a final readout under immunofluorescence microscopy (Leica, UK). The controls included mock-infected and/or mock-treated wells.

Cells were fixed with 4% formaldehyde for 20 min, permeabilized with 0.3% Triton X-100 for 10 min, blocked with 5% bovine serum albumin, and incubated with a primary antibody recognizing HuR (1:50) and HIF-1α (1:50). Secondary antibodies were used to detect primary antibody-antigen complexes with Cy3 (Abcam, Cambridge, UK). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min in a dark room. Finally, images were detected by immunofluorescence microscopy (Leica, Wetzlar, Germany).

Vero E-6 and MA-104 cells cultured in 24-well-plates were infected with an MOI at an 0.1 (passage 4 virus). The cultures were harvested by scraping cell monolayers from at different time points (6, 12, 18, 24, 48 and 72 h) and stored at -80°C. The Vero E-6 cells were then inoculated with 10-fold serial dilutions of the samples in triplicate per dilution. The viral inoculum was removed. The cell monolayers were overlaid with virus medium containing 1% CMC. The cells were fixed with 10% neutral buffered formaldehyde after infection of 72 h at room temperature for 20 min, and permeabilized with Triton X-100. The wells were then incubated with a human antibodt to the SARS-CoV-2 nucleocapsid protein (1:2500) (GenScript; HC2003) for 1h in TBST at 37°C and washed 3 times with TBST. The cells were incubated for 1 h with goat anti-human IgG conjugated to fluorescein isothiocyanate (FITCH) (Southern Biotech, USA) and diluted 1:1000 in TBST and then washed three times with TBST and once with distilled water. Antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (Leica, UK). The fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units per ml.

Brain tissues were trimmed, and tissues containing the substantia nigra pars compacta were sectioned into 20 μm slices (Leica, Germany). Fifty frozen sections from each mouse were divided into four sets. One set was randomly selected for immunofluorescence staining to observe the colocalization of GHS-R1a and D₂R. Brain slices and cells were blocked with 10% goat serum and incubated with mouse anti-GHS-R1a (Absin, 1: 50) and rabbit anti-D₂R (Santa Cruz, 1: 50) overnight at 4 °C. The cells or tissues were then washed with PBS and incubated with Alexa Fluor® 555-conjugated goat anti-mouse IgG (H + L) (1:500), Alexa Fluor® 488-conjugated goat anti-rabbit IgG (H + L) (1:500) and Alexa Fluor® 555-conjugated goat anti-rabbit IgG (H + L) (1:500) secondary antibodies for 1 h at room temperature, and DAPI was added for 5 min before mounting with antifade solution. Images were acquired using immunofluorescence microscopy (Leica, Germany).