Pegfr

Followed by euthanization of mice and PBS perfusion through the left ventricle, VAT and liver were excised and fixed with 4% buffered formalin for 2 hours and stored in 30% sucrose solution containing 0.05% sodium azide for overnight. Then, the tissues were embedded in OCT blocks and cut into sections (6 μm). Samples were permeabilized with 0.1% triton X-100 for an hour. After blocking, mouse tissue sections were stained with anti-F4/80 (Invitrogen, #MA1–91124; Thermo Fisher, MA5–16363), CD11b (Abcam, #ab133357), CD31/VE Cadherin (Abcam, #ab7388; Thermo Fisher, #MA1–80069), VCAM-1 (Invitrogen, #PA5–86042), p-EGFR (Invitrogen, #44–794G), and MCP-1 (Invitrogen, #MA5–17040) followed by staining with fluorochrome conjugated secondary antibodies. The sections were stained and fixed with Vectashield mounting medium with DAPI (Vector Laboratories, #H 1200), and images were taken using Nikon A1 spectral confocal for immunofluorescence scanning using 20X magnification. Livers from 4–5 mice per group were sectioned 3 times, and 2 images per section were taken. Image analysis was done using ImageJ software. Distance measurement of macrophages from sinusoidal EC was performed by ImageJ by drawing straight lines from macrophages to the nearest sinusoidal EC.