Alexa fluor 488 conjugated isolectin gs ib4

We measured CNV volume as previously described (Mao et al., 2017 (link); Wei et al., 2018 (link); LeBlanc et al., 2019 (link)). In brief, 7 days after laser photocoagulation, we sacrificed the mice and enucleated the eyeballs. After fixing the eyeballs in 4% paraformaldehyde (Electron Microscopy Sciences [EMS], Hatfield, PA) for 1 h, we removed the corneas, the lenses, the vitreous, and the neurosensory retinas. The choroid/RPE/scleral tissue was isolated with four radial incisions and washed three times in PBS. Then we incubated the processed choroid/RPE/scleral complexes with blocking buffer (PBS containing 1% BSA and 0.5% Triton X-100) for 4 h at room temperature. After washing three times in PBS, the RPE complexes (RPE/choroid/sclera) were stained with 10 μg/ml Alexa Fluor^®488 conjugated isolectin GS-IB4 (Invitrogen, Cat. No. 121411) in a volume of 0.5 ml/retina overnight at 4°C and flat-mounted with antifade mounting medium (VECTASHIELD; Vector Laboratories, CA) to preserve the fluorescence. We observed CNV with a Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss) at a magnification of 100×. An average of 3–4 CNV areas per eye was analyzed. Each CNV spot was considered an independent data point. For each group, at least 25 spots were analyzed from eight mice. All quantifications were conducted under blinded conditions.

The newborn C57BL/6J mice and their nursing mothers were placed in a 75% oxygen supply chamber from P7 to P12, and then were returned to room air for 5 days. The mice received an intravitreal injection of 1 μL of 10% DMSO (Ctrl), 1 μL of DCZ19931 (1 μg/μL), 1 μL of Ranibizumab (10 mg/mL), or 1 μL mixture solution of DCZ19931 (1 μg/μL) plus Ranibizumab (10 mg/mL) immediately when returning to room air at P12. They were anesthetized and the eyeballs were removed at P17. The retinas were stained with Alexa Fluor 488-conjugated isolectin GS-IB4 (1:100, Invitrogen, I21411, USA).

Animals were transcardially perfused with 4% paraformaldehyde. Lumbar DRG L4 and L5 were dissected, cryoprotected in 20% sucrose, mounted in OCT media, and sectioned at a thickness of 12 μm. Slides were washed with 1× PBS thrice (2 min for each wash). The sections were blocked with 0.2% Triton X-100 in 10% NGS for 30 min. Sections were washed with 1× PBS twice (2 min for each wash). Sections were incubated with primary antibodies diluted in a blocking buffer overnight in a humid chamber (Anti-CGRP, Peninsula labs, San Carlos, CA, United States). After the incubation of primary antibodies, sections were washed with 1× PBS three times (2 min for each wash) and then incubated the sections with secondary antibodies (1:1000) and Alexa Fluor 488-conjugated Isolectin GS-IB4 (Thermo Fisher Scientific, Waltham, MA, United States) in a humid chamber. Finally, the sections were washed with 1× PBS five times (5 min for each wash). For counting, L3 and L4 DRG pairs from three different animals were counted.