Cd31 nb100 2284

F344 rats (n = 4) were implanted with 2 NICHE (1 in each flank), loaded with 5 × 10⁵ BM-MSCs and vascularized for 6 weeks. For each rat, one NICHE drug reservoir was loaded with 9 mg CTLA4Ig (30 mg/mL) and 0.6 mg ALS (2 mg/mL) for local Immunosuppressant (IS) release NICHE. The contralateral NICHE drug reservoir was loaded with saline and served as a control. Two weeks later, NICHE were harvested and processed for histology and VEGF quantification. For functional blood vessel staining, following pressure-cooker-based epitope retrieval, sections were stained with primary antibodies CD31 (NB100-2284, Novus Biologicals, 1:200), VE Cadherin (36-1900, Invitrogen, 1:25), and eNOS (ab300071, Abcam, 1:50). Anti-rabbit Alexa Flour 555 (A-21428, Invitrogen, 1:200) was used as secondary antibody. Imaging was performed using a Nikon Eclipse TE300 Fluorescent Microscope in the FL2 channel (585 ± 20 nm). Fluorescence intensity was measured by 2 independent observers using the NIS-Elements Basic Research software. VEGF was quantified from tissue lysate using Bio-Plex Pro Rat Cytokine VEGF Set (#171L1026M) magnetic bead-based assay (Bio-Rad Laboratories) on the Bio-plex platform (Bio-Rad), according to manufacturer’s instructions.