Immunohistochemistry of murine retina

Immunohistochemistry of murine retinas (mCD31/hCD31)

Mice were anesthetized by intramuscular injection of ketamine/xylazine and perfused via intracardiac administration of 10 ml of 1× PBS, followed by 10 ml of 4% paraformaldehyde in PBS. Eyes were enucleated, immersion-fixed overnight in 4% paraformaldehyde in PBS at 4°C, and stored in PBS until processing. For retinal sections, eyes were placed in 30% sucrose in PBS overnight at 4°C before preparation of the frozen blocks in optimal cutting temperature embedding medium (Fisher HealthCare). Serial cross sections of retinas (20 um) were cut and mounted on slides. Tissue sections were stored at −80°C until ready to be processed. Samples were allowed to thaw for a minimum of 1 hour at room temperature. Samples were then fixed in ice-cold acetone for 5 min and let dry for 10 min at room temperature. A 15-min permeabilization step in tris-buffered saline (TBS)–0.05% Tween 20 (TBS-T) followed. Antigen retrieval was then performed in 1× sodium citrate buffer (Vector Laboratories Inc.) for 30 min at 95°C. After a 30-min cool down step, to further reduce background staining, samples were incubated in 1% Triton X-100 in TBS for 30 min at room temperature. To block nonspecific binding, samples were incubated in 10% donkey serum in TBS for 30 min at room temperature. Following three washes in TBS-T (2 min each), retinal sections were incubated for 10 min with 3% H2O2 in TBS to block potential endogenous peroxidase activity. After another round of washes, tissue sections were reacted with primary antibodies overnight at 4°C: FITC-conjugated mouse anti-hCD31 (1:25; Santa Cruz Biotechnology, sc-53411), rat anti-mouse CD31 (1:25; BD Biosciences, 550274). Following three washes in TBS-T (2 min each), retinal sections were incubated with donkey anti-rat DyLight 594 (1:250; Thermo Fisher Scientific, SA5-10028) for 1 hour at room temperature. After incubation with secondary antibodies, samples were extensively washed and then incubated with DAPI (Thermo Fisher Scientific) for nuclear staining for 10 min at room temperature. Last, the retinal tissue was washed with PBS and mounted with the VECTASHIELD Hardset Antifade Mounting Medium (Vector Laboratories Inc.). Digital images were acquired at the UAB High Resolution Imaging Facility core using a Zeiss microscope (Axio Imager 2, upright microscope) and a Nikon A1R confocal microscope. The system was operated by Nikon NIS Elements 5.21 software. Z-stack images were acquired at 1024 by 1024 pixel density denoised with NIS.ai algorithm and saved as 3D reconstructed files.