P0262

The brains (n = 4/group) embedded with OCT were manufactured into 10-μm-thick frozen sections of TNC tissue in a cryostat microtome (Leica 1950 M). After fixation in ice-cold acetone, the sections were blocked by blocking buffer (10% normal goat serum, 0.5% Triton X-100, dissolved in 0.1 M PBS) for 1 h at room temperature (RT). Then, the sections were incubated with primary antibody (rabbit anti-GFAP antibody, 1:100, ab207165, Abcam) diluted in primary antibody dilution buffer (P0262, Beyotime) overnight at 4 °C. After cleaning with 0.1 M PBS three times, the sections were incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG, 1:1000, ab150077, Abcam) diluted in secondary antibody dilution buffer (P0265, Beyotime) for 1 h at RT. Then, the sections were rinsed in 0.1 M PBS three times and mounted with an antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, P0131, Beyotime). Magnified Images (× 20 objective) were captured under a fluorescence microscope (BX43, Olympus) using the cellSens standard software (version 1.18, Olympus). In negative-control sections, PBS was used instead of primary antibody, and there were no positive signals. The immunofluorescence area fraction was measured using ImageJ software (version 1.52p, National Institutes of Health).

Followed by three cycles of washing with PBS, 5 min each, 0.5% TritonX-100 was provided dropwise to slides and let stand by for 10 min at room temperature. The slides were immersed in PBS 3 times, 3 min each, blocked by using 10% normal goat serum (C0265; Beyotime) at room temperature, and removed after blocking for 60 min. The primary antibody diluent (P0262; Beyotime) was supplemented, placed in a humid chamber, and incubated at 4°C overnight. The slides were rewarmed at room temperature for 30 min and soaked in PBS for 3 times, 3 min each. Excess liquid was removed, and fluorescent secondary antibody was diluted and added for incubation at 37 °C for 60 min. PBS immersion was conducted for 3 times again, 3 min each. Following the addition of DAPI (C1005; Beyotime), the cells were incubated in the dark for 5 min and stained for nucleus. Antifluorescence quenching medium was added to mount the slides, and the experimental results were collected using fluorescence microscope imaging system (MF53; Guangzhou Mshot Photoelectronic Technology Co., Ltd.).

Guinea pigs were sacrificed by cervical dissection. After removing muscle and anterior segments of the eye, eyecup was fixed using 4% paraformaldehyde (P0099, Beyotime, China) for 0.5 h, followed by impregnating using 10% sucrose in PBS for 2 h, 20% sucrose for 2 h, and then 30% sucrose for 15 h, and finally embedding into Optimal Cutting Temperature (OCT) compound to freeze immediately with liquid nitrogen. The embedded tissue was sectioned into 10 μm thickness for immunofluorescence analysis. Retina slides were blocked with QuickBlock™ solution (P0260, Beyotime, China) for 1 h, then the primary antibodies (Mice anti-SNCA, 1:500, AHB0261, Invitrogen, United States; Rabbit anti TH, 1:1,000, AB152, Sigma-Aldrich, United States) were diluted with a blocking solution (P0262, Beyotime, China) and used to incubate retinas for 16 h at 4°C. After incubating and rinsing, secondary antibodies conjugated to Goat anti-Rabbit-Cy3 (1:1,000, A0516, Beyotime, China) and Goat anti-Mice-Alexa Fluor 488 (1:1,000, ab150113, Abcam, United States) were applied for 2 h at room temperature. Zeiss LSM800 (Carl Zeiss, Germany) and a confocal microscope was used to take micrographs at 10 × 20 fold.