Immunohistofluorescence (IHF)

iWAT was harvested from WT and KO mice, fixed with 4% PFA, and embedded in paraffin. The embedded tissues were cut into 5 μm-thick sections and stained with H&E. To quantify the blood vessel density, all sections were photographed under a microscope. The blood vessels were categorized into large vessels and capillaries according to their sizes. The large vessel and capillary densities were presented as the number of those per photographed image.

To determine the presence and expression of CD31 in adipose tissue, 4-μm sections of specimens were cut from formalin-fixed paraffin-embedded (FFPE) blocks. Slides were heated for at least one hour in a dry oven at 60℃, then followed by multiplex immunofluorescence staining with a Leica Bond Rx Automated Stainer (Leica Biosystems, Newcastle, UK). Briefly, the slides were baked for 30 min and dewaxed with Leica Bond Dewax solution (#AR9222, Leica Biosystems), followed by antigen retrieval with Bond Epitope Retrieval 2 (#AR9640, Leica Biosystems) in a pH 9.0 solution for 30 min. The primary antibodies for CD31 (ab124432, Abcam, dilution 1:500) were incubated for 30 min in a humidified chamber at room temperature, followed by detection using the Polymer HRP Ms + Rb (ARH1001EA, AKOYA Biosciences) for 10 min. Visualization of CD31 was accomplished using Opal 690 TSA (dilution 1:150) for 10 min, after which the slide was treated Bond Epitope Retrieval 1 (#AR9961, Leica Biosystems) for 20 min to remove bound antibodies before the next step in the sequence. Nuclei were subsequently visualized with DAPI, and the section was coverslipped using ProLong Gold antifade reagent ({"type":"entrez-protein","attrs":{"text":"P36934","term_id":"549428","term_text":"P36934"}}P36934, Invitrogen). To quantify the CD31 staining area, all sections from three mice per experimental group were selected. The immunostaining area of CD31 was measured using ImageJ software, as previously described⁴⁶. The red channel was separated from blue, converted to greyscale, and the background was subtracted. The values of the monochromatic immunofluorescent stacks were then measured and expressed as means of area percentage.