Alexa fluor 594 488 conjugated goat anti mouse rabbit secondary antibody

For immunohistochemistry, eyes were removed from euthanized mice by intraperitoneal injection of pentobarbital (75 mg/kg) and by cervical dislocation and fixed in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 1 h at 4 °C, followed by cryoprotection in 30% sucrose for 2 h. The lenses were removed, and the eyes were embedded in optimal cutting temperature (OCT) solution and sectioned at a 10-μm thickness. After blocking and permeabilization with 10% normal donkey serum and 0.2% Triton X-100 in phosphate buffer for 1 h, the sections were labeled with the primary antibody overnight at 4 °C. The primary antibodies used are shown in Additional file 4: Table S3. The sections were rinsed in PBS three times, Alexa Fluor 594/488-conjugated goat anti-mouse/rabbit secondary antibody (Cat# A11005 and A11008, Invitrogen, Waltham, MA, USA, 1:500 dilution) was applied, and nuclei were counterstained with DAPI (Cat# D8417, Sigma, St Louis, MO, USA) for 1 h at room temperature. Images were captured on a Zeiss LSM 800 confocal scanning microscope. In addition, the fluorescence intensity was quantified using ZEN 2.3 imaging software.

For immunohistochemistry, eyes were removed from euthanized mice by intraperitoneal injection of pentobarbital (75 mg/kg), and by cervical dislocation and fixed in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 1 h at 4 °C, followed by cryoprotection in 30% sucrose for 2 h. Lens were removed and eyes were embedded in optimal cutting temperature solution (OCT) and sectioned at 10 μm thickness. After blocking and permeabilization with 10% normal donkey serum and 0.2% Triton X-100 in phosphate buffer for 1 h, the sections were labeled with the primary antibody at 4 °C overnight. The primary antibodies used are shown in Table S3. The sections were rinsed in PBS three times and Alexa Fluor 594/488-conjugated goat anti-mouse/rabbit secondary antibody (Cat# A11005 and A11008, Invitrogen, Waltham, MA, USA, 1:500 dilution) was applied and nuclei were counter-stained with DAPI (Cat# D8417, Sigma, St Louis, MO, USA) for 1 h at room temperature. Images were captured on a Zeiss LSM 800 confocal scanning microscope. In addition, the expression levels of CD68 and GFAP were compared between WT and mutant retinas by quantifying their fluorescence intensities using ImageJ software.

COS-7 cells were seeded in 24-well plates (Corning, Corning, NY, USA) and transfected at 70% confluency with constructed vectors or empty vectors using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. Cells were harvested after 48 h and fixed in 4% paraformaldehyde for 15 min at room temperature. After blocking with 1× PBS containing 5% normal goat serum and 0.2% Triton X-100, cells were incubated with specific antibodies at 4 °C overnight. The primary antibodies used are shown in Table S3. AlexaFluor 594/488-conjugated goat anti-mouse/rabbit secondary antibody (Cat# A11005 and A11008, Invitrogen, Waltham, MA, USA, 1:500 dilution) was applied and nuclei were counter-stained with DAPI (Cat# D8417, Sigma, St Louis, MO, USA).