T inverted

Primary cells were isolated from 26 days PW pooled wound tissues of LysM-Cre;tdTomato mice. Tissues were micro-dissected, minced, and incubated in a Collagenase IV solution for 60 min with constant rotation. Post-incubation, cells were filtered through a 40 µm nylon screen, centrifuged, washed, resuspended in DMEM supplemented with 10% FBS, antibiotics, and antifungals and cultured at 37 °C in a water-jacketed incubator with 5% CO₂ output. Once cells reached confluency, they were switched to adipocyte differentiation media (Cell Solutions) for 72 h, upon which they were switched to adipocyte maintenance media (Cell Solutions) for additional 7 days. Cells were incubated with BODIPY (Thermo) as per manufacturer’s directions and visualized using a Nikon T inverted (Nikon) microscope.

Tissues were fixed in 4% PFA, dehydrated, paraffin embedded, and sectioned at 7–12 µm thickness. Frozen tissues were sectioned at 12 µm thickness. Immunostaining was performed both on frozen and paraffin sections. Heat-based antigen retrieval was performed when necessary. Tissue sections were blocked in either 3% BSA, 3% Donkey serum, or 3% mouse IgG from Mouse on Mouse (MOM) kit (Vector Labs). The primary antibodies used were goat anti-FABP4 (1:200; R&D Systems), mouse anti-SMA (1:500; R&D Systems), rabbit anti-RGS5 (1:250; Proteintech), rabbit anti-LYZ (1:250; Abcam), rabbit anti-CRABP1 (1:800; Cell Signaling), goat anti-CYP26B1 (1:50; Novus Biologicals); rabbit anti-G0S2 (1:100; Proteintech), mouse anti-MEST (1:250; Proteintech), goat anti-PDGFRA (1:350; R&D Systems), chicken anti-β-Gal (1:500; Abcam), and rabbit anti-PLIN (1:800; Cell Signaling). Tissue sections were visualized with Nikon T inverted (Nikon) and Olympus FluoView confocal (Olympus) microscopes.