Eyes of normal and hyperoxia-exposed P12, P15, and P18 mice were dissected after cardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and fixed for 4 hours at 4°C. Fixed eye cups were infiltrated overnight with 30% sucrose in the same buffer, embedded in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC), and cryosectioned at 10 μm. For immunostaining, retinal sections were pretreated with a blocking solution (1% normal goat serum, 1% bovine serum albumin, and 0.05% Triton X-100 in PBS [pH 7.4]) for 1 hour at room temperature and then incubated with primary antibody (STAT3 antibody or p-STAT3 antibody) diluted 1:100 in PBS with 0.3% Triton X-100 overnight at 4°C. After PBS rinse, the sections were treated with Cy3-conjugated goat anti-rabbit IgG (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA), in PBS for 1 hour at room temperature. Slides were then washed in PBS and incubated in fluorescein Griffonia simplicifolia lectin, isolectin B4 (Vector Laboratories) for 20 minutes, washed in PBS, and mounted (Fluoromount-G; Southern Biotechnology Associates, Birmingham, AL). Preabsorption tests were performed by preincubating the diluted anti-STAT3 antibody with blocking peptide for 1 hour before the immunostaining procedures. Sections were viewed with a microscope equipped for epifluorescence (model TE300; NikonUSA, Melville, NY). Images were captured with a digital camera (Spot; Diagnostic Instruments, Sterling Heights, MI). Some sections were also evaluated with confocal microscopy (model LSM510; Carl Zeiss Meditec, Dublin, CA).