Ab60946

Paraffin-embedded gWAT sections were used for routine histology. Deparaffinized tissue slides were stained with H&E for morphological structure and PAS stain for ECM. Adipocyte size was assessed using light microscopy and quantified through Image-Pro Premier 9.2 software.
Antibodies targeting the proteins EDEM3 (1:100, NBP1-88342, Novus Biologicals, Centennial, CO, USA), VEGFB (1:200, MAB751, R&D Systems, Minneapolis, MN), UCP-1 (1:1,000, ab10983, Abcam, Waltham, MA), p-AKT (1:50, 4060, Cell Signaling Technology), and p-INSR (1:50, ab60946, Abcam) were used in IHC. Biotinylated goat anti-rabbit (1:400, BA-1000, Invitrogen) was used as a secondary antibody. Images were taken at 20× magnification, and InDen was quantified using ImageJ software.
Antibodies for other proteins characterized by specific IF included anti-F4/80 (1:200, 14-4801-82, Invitrogen), anti-EDEM3 (1:100, NBP1-88342, Novus Biologicals), anti-CD31 (1:20, ab28364, Abcam), anti-VE-Cadherin (1:150, ab33168, Abcam), and anti-Perilipin (1:300, 20R-PP004, Fitzgerald Industries). Secondary antibodies employed were Alexa Fluor 488, goat anti-rabbit (1:400, A11034, Invitrogen), and Alexa Fluor 555, goat anti-mouse (1:400, A21422, Invitrogen). Images were taken at 20× magnification (KEYENCE-BZ-×800 series, Osaka, Japan), and fluorescence intensity was measured using ImageJ software (ImageJ.win32).

HUVECs were fixed in 4% paraformaldehyde at 4°C for 15 min and incubated in hydrogen peroxide for 15 min. Subsequently, the cells were blocked with goat serum (Solarbio) at 37°C for 30 min, followed by incubation with anti-IKK (1:100, ab32041; Abcam), anti-IKB-α (1:100, ab32518; Abcam), anti-NALP3 (1:150, 19771-1-AP; ProteinTech Group, Inc.), anti-NFκB (1:150, 14220-1-AP; ProteinTech Group, Inc.), anti-p-INSR (1:100, ab60946; Abcam), anti-p-ISR-1 (1:100, ab3690; Abcam) and anti-TLR4 (1:200, ab22048; Abcam) at 4°C overnight.