Alexa fluor 488 conjugated goat igg secondary antibody

Immunohistochemical staining of 7-μm frozen sections of full-thickness retina was prepared. Three sections per frozen block from each group (n = 3 retinas/group) were used for immunohistochemical analysis. Tissue sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature and then washed thrice with PBS. Sections were next blocked with 5% bovine serum albumin/PBS for 1 h at room temperature and then with the primary antibodies against monoclonal mouse anti-GFAP antibody (1:200; Abcam), polyclonal rabbit anti-parkin (1:200; Abcam), or polyclonal rabbit anti-optineurin (1:50; Abcam) for 16 h at 4 °C. After several washes, the tissues were incubated with Alexa Fluor 488-conjugated goat IgG secondary antibody (1:200; Life Technologies) for 1 h at room temperature and then washed with PBS. The sections were counterstained with Hoechst 33342 (1 μg/ml; Life Technologies) in PBS. Images were captured by a confocal microscopy (Leica SP8).

Immunohistochemical staining of 7-um frozen sections of full-thickness retina was prepared. Three sections per frozen block from each group (n = 3 retinas/group) were used for immunohistochemical analysis. The RGCs (n = 3 per group) on the coverslips were fixed with 4% PFA in PBS for 20 min, rinsed with PBS, and then were used for immunohistochemical analysis. The samples were permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature, sequentially, blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, incubated by 16 h at 4°C with the following primary antibodies: monoclonal mouse anti-GFAP antibody (1:200; Abcam), polyclonal rabbit anti-OPA1 (1:200; Abcam), and monoclonal rabbit anti-LC3 (1:1000; Abcam) for 16 h at 4°C. After several washes, the samples were incubated with Alexa Fluor 488-conjugated goat IgG secondary antibody (1:200; Life Technologies) and Hoechst 33342 (1 μg/mL; Life Technologies). Fluorescence imaging and analyses were performed by a confocal microscopy (Leica SP8).