Anti p16ink4a antibody

The cells were placed on glass slides, fixed in acetone for 5 min at room temperature (15–25° C), and dried completely before staining. Cells were incubated overnight at 4° C with an anti-SFRP4 antibody (Thermo Fisher Scientific) and anti-p16^ink4a antibody (Abcam, Cambridge, UK) diluted at 1:100 in phosphate-buffered saline (PBS). After washing thrice with PBS, the slides were incubated with Alexa Fluor 488 conjugated goat anti-rabbit antibody and AlexaFluor555 conjugated donkey anti-goat antibody (Thermo Fisher Scientific) diluted at 1:2000 in PBS for 1 h at room temperature. After incubation, the slides were washed thrice with PBS and counterstained for nuclear visualization using ProLong Gold Anti-fade Mountant (Thermo Fisher Scientific) containing 4ʹ,6-diamidino-2-phenylindole.

The isolated retinal vasculature was snap frozen in optimal cutting temperature (OCT) solution. Cryostat sections (10 μm) were obtained, mounted on glass slides and permeabilized with 1% Triton for 10 min and blocked in 10% normal goat serum for 1 h. Sections were then incubated with anti-p16^INK4A antibody (Abcam, Cambridge, MA, USA; Cat. # ab189034; 1:50) and Alexa Fluor 594-conjugated isolectin GS-IB4 (Invitrogen, Carlsbad, CA, USA; Cat. # I21413; 1:200) at 4 °C overnight. On the next day, the sections were washed three times with phosphate-buffered saline (PBS) and were then incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA; Cat. # A11034; 1:400) that was washed in PBS, and covered with mounting medium containing 4′,6′-diamino-2-phenylindole (DAPI) for counterstaining (Vectashield; Vector Laboratories, Burlingame, CA, USA; Cat. # H-1200). Images from three to four samples per group were taken using Zeiss Axioplan2 Imager Microscope (Zeiss, San Diego, CA, USA). The results were analyzed by measuring the fluorescence intensity of p16 immunolabeling using ImageJ software.