Whole-mount Tissue Staining Protocol

Whole-mount tissues were stained according to our previously published methods^{47 (link)–49 (link)}. Briefly, tissues were fixed at 4 °C with 4% PFA in PBS overnight, washed with PBS, and cut into small pieces. Tissues were digested with proteinase K (20 mM) and permeabilized with 100% methanol. Samples were blocked overnight with a PBS-0.1% Triton X-100 solution containing 3% milk, and incubated at 4 °C overnight with primary antibodies against CD31 (1:100, Cat. No. AF3628, R&D) and Perilipin (1:200, Cat. No. 20R-PP004, Fitzgerald Industries). After washing with PBS, samples were blocked with 3% milk, and incubated at RT with fluorescent-conjugated secondary antibodies (1:300, Thermo Fisher Scientific Inc.) for 2 h. After rigorous rinsing, samples were mounted with a Vectashield mounting medium (Cat. No. H-1000, Vector Laboratories). Fluorescent signals were examined under a confocal microscope (LSM510 Confocal, Zeiss).