Alexa fluor 488 conjugated ib4

On d 7 after CNV, after the removal of the anterior portion, radial relaxation incision was made in the eyecup of the sclera/choroid/RPE complexes. Then immunostaining was performed in choroidal flat mounts. The choroidal tissues were incubated with a 1 mg/mL solution of Alexa Fluor 488‐conjugated IB4 (#I21411, Thermo Fisher Scientific) and Alexa Fluor 594‐conjugated collagen type I (#bs‐10423R‐A594, Bioss Inc) antibodies at 4°C for 24 hours.

Cells were harvested and counted as described previously [15 (link)] with some modifications. Briefly, two retinas were minced in 1 ml of Hank’s Balanced Salt Solution (Thermo Fisher Scientific) containing 2 mg/ml Collagenase D and 28 U/ml DNase I (both from Sigma-Aldrich). Tissue was incubated at 37 °C for 60 min, and re-suspended in 2 ml of Hibernate media (Thermo Fisher Scientific). The suspension was filtered through a 140 μm-mesh screen (Sigma-Aldrich), washed, and centrifuged; supernatant was carefully removed. Cell pellets were fixed and permeabilized for 10 min at RT in 4% paraformaldehyde/0.1% saponin buffer (Sigma-Aldrich), then blocked for 15 min at RT in 3% BSA. Cells were incubated in Alexa Fluor 647 conjugated anti-mouse CD11b antibody (1:100, Biolegend, USA) and Alexa Fluor 488 conjugated IB4 (1:100, Thermo Fisher Scientific) overnight at 4 °C. Suspensions were finally washed and re-suspended in 300 μl PBS. Data were acquired on a BD FACScan flow cytometer (BD Biosciences), 10,000 events were collected for each sample and then analyzed using the FlowJo software (Tree Star).

As previously reported [3 (link)], after euthanasia, whole eyes from mice on postnatal day (P) 6.5 were fixed in 4% paraformaldehyde for 1 h at room temperature (RT). After blocking and permeabilization in retina-blocking buffer (1% bovine serum albumin [BSA] and 0.5% Triton X-100 in PBS) overnight, the retinas were incubated at 4 °C in PBLEC buffer (1 mM CaCl₂, 1 mM MgCl₂, 0.1 mM MnCl₂, and 1% Triton X-100 in PBS) with Alexa Fluor 488-conjugated IB4 (Thermo Fisher, I21411, 1:200) overnight. After washing with PBLEC buffer, the retinas were dissected into four leaflets. The retinas were examined by confocal laser microscopy (Zeiss LSM 900 with Airyscan2).