Fluorescence conjugated secondary antibody

Cells were fixed with 4% PFA for 15 min, RT, washed with PBS three times, then permeabilized with 0.3% PBSTr (Triton-X100) for 15 min. Permeabilization is not required for cell surface antigens. After blocking in 3% BSA for 30 min, the cells were incubated with primary antibodies at 4 °C, overnight. The next day, after washing with 0.1% PBST (Tween) three times, the cells were incubated with fluorescence-conjugated secondary antibodies (Abcam, 1:200) for 1 h, RT, then washed with 0.1% PBST three times. The nuclei were stained with DAPI (Sigma-Aldrich, 1:10000). Images were captured with fluorescence microscope (Leica, Germany). The primary antibodies used were: anti-SSEA-4 (Santa Cruz, sc59368, 1:200), anti-OCT4 (Abcam, ab181557, 1:200), anti-TBXT (R&D systems, AF2085, 1:200), anti-CX43 (Sigma-Aldrich, C6219, 1:400), and anti-NR2F2 (Santa Cruz, sc393481, 1:100).

Prepared frozen sections of tumors were respectively incubated with anti-mouse CD31 antibody (BD Biosciences, Franklin Lakes, NJ, USA) and anti-human PEDF antibody (R&D Systems, Minneapolis, MN, USA) overnight, and subsequently with a fluorescence-conjugated secondary antibody (1:100; Abcam, Cambridge, MA, USA) for 45 min. The CD31-positive vessels and the PEDF-positive reaction were visualized with 3,3′-diaminobenzidine (DAB; ZSJQ Biotechnology).

Cells were cultured in a 12-well plate. Microtubule-associated protein 1 light chain 3 (LC3; 14600-1-AP, ProteinTech, Wuhan, Hubei, China) and activated β-catenin were detected using a fluorescence microscope (EU5888; Leica). Briefly, after treatment, cells were fixed in 4% paraformaldehyde for 15 min. Next, they were blocked in 5% bovine serum albumin for 30 min and incubated overnight with anti-LC3 (1:400; Cell Signaling Technology, Shanghai, China) and activated β-catenin (1:100; Cell Signaling Technology) at 4°C. The next day, cells were washed with phosphate-buffered saline and incubated with a fluorescence-conjugated secondary antibody (Abcam, Shanghai, China) for 2–3 h at 37°C, and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Shanghai, China) for 2 min. Cells were observed using an inverted fluorescence microscope (Leica).

Choroidal flat mount preparation, staining, and imaging were undertaken as previously described [21 (link)]. Briefly, eyes were enucleated at 7 day after laser injury and immediately fixed for 1 h in a solution of 4% paraformaldehyde in phosphate-buffered saline (PBS; 9 g/L NaCl, 0.232 g/L KH₂PO4, and 0.703 g/L Na²HPO4; pH 7.3). The anterior segment and crystalline lens were removed, and the retinas were detached and separated from the optic nerve head with a pair of fine-curved scissors. The remaining eye cups were washed with cold ICC buffer (0.5% BSA, 0.2% Tween 20, and 0.05% sodium azide) in PBS. Next, a 1 : 500 dilution of isolectin B4 (Sigma-Aldrich, USA) and 1 : 1000 dilution of CD31 (Abcam, Cambridge, UK) were incubated at 4°C overnight and then washed with cold PBS buffer. A 1 : 1000 dilution of fluorescence-conjugated secondary antibody (Abcam, Cambridge, UK) was incubated for 1 hour and then washed with cold PBS buffer. Radial cuts were made toward the optic nerve head, and the sclera-choroidal/RPE complexes were flat mounted, covered, and sealed. Image J software was used to analyze fluorescence images. The summation of the whole stained area in each section multiplied by the distance between sections (1 μm) was used as an index for the CNV lesion volume. The volumes of the all lesions in each eye were averaged and considered as an n = 1 for statistical analysis.

Immunofluorescence staining was performed as previously described [16 (link), 18 ]. Briefly, after the section was repaired, the slices were incubated with 5% BSA at room temperature for 30 min. The slices were incubated at 4 °C overnight with the following primary antibodies: Sirt1 antibody (1:100, bs-0921R, Bioss). Then, the slices were incubated with the appropriate fluorescence-conjugated secondary antibody (1: 500, Abcam, MA, USA) at 37° C for 1 h.