Cy3 labeled donkey anti rabbit igg

The identity of Müller cells was confirmed by immunocytochemical analyses using antibodies specific for Müller cells, GS, and glial fibrillary acidic protein (GFAP). Briefly, the cells were fixed with 4% paraformaldehyde for 20 min. Then, the samples were incubated with 0.3% Triton X-100 for 10 min and blocked with 1% bovine serum albumin (BSA) for 30 min. Afterwards, the cells were treated with the following primary antibodies overnight at 4°C: rabbit monoclonal anti-GS (1 : 5000; Abcam) or mouse polyclonal anti-GFAP (1 : 200, Abcam). On the following day, the samples were further incubated with the following secondary antibodies: Cy3-labeled donkey anti-rabbit IgG (1 : 200, Biolegend) or FITC-labeled donkey anti-mouse IgG (1 : 200, Jackson ImmunoResearch, PA, USA), for 2 hours at room temperature. Then, the cells were washed again three times for 10 min each with PBS and stained with 1 μg/mL DAPI (1 : 100, Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Afterwards, the immunoreactive fluorescence staining of the cells was photographed using a Zeiss Imager M1 laser scanning microscope (Carl Zeiss, Germany).

Frozen sections were fixed with 4% neutral buffered paraformaldehyde (Bmassay, Beijing, China) for 10 min and then blocked with PBST containing 1% BSA for 2 h at room temperature. After blocking, the sections were incubated with primary antibody including NPR1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:400, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:200, Proteintech, Wuhan, Hubei, China), and CD31 (1:100, R&D, Minneapolis, MN, USA) at 4 °C overnight. Subsequently, the sections were incubated with secondary antibody Cy3-labeled donkey anti-rabbit IgG (1:200, Biolegend. San Diego, CA, USA) or Cy5-affinipure donkey anti-goat IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) at room temperature 1 h. The nucleus was stained with Hochest3342 (Beyotime, Shanghai, China). The sections were examined by confocal microscope (Carl Zeiss, Oberkochen, Germany).