488 conjugated goat anti mouse secondary antibody

Adipose tissues were stained with hematoxylin and eosin (HE) according to the standard protocols. For immunohistochemistry, rabbit anti-UCP1 antibody (Alpha Diagnostic, ucp1-a, diluted 1:400) or rabbit anti-IRX3 (Abcam, ab25703, diluted 1:1000) was used to incubate the sections, followed by incubation with HRP-linked secondary antibody (Dako, k500711). Electron microscopy of browning adipose tissue was performed as previously described (Wang et al., 2013 (link)). To determine the localization of IRX3 in the browning adipocytes, beige cells after eight days induction were fixed and incubated with rabbit anti-IRX3 antibody (Abcam, ab25703) and mouse anti-UCP1 antibody (R&D, MAB6158), followed by incubation with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (ThermoFisher, R37117), 488–conjugated goat anti-mouse secondary antibody (ThermoFisher, A32723), and DAPI (SouthernBiotech, 0110-20). Images were acquired with a confocal laser-scanning microscope (Zeiss). All the images were representative of three independent experiments.

Cells were seeded on glass coverslips in a 24−well plate. The attached cells were then treated with 800 μM PQ or PQ-Biotin for 24 h as indicated, fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 (Biosharp, BS100) for 6 min at room temperature. The permeabilized cells were then blocked with 0.2% BSA for 30 min and incubated with primary antibodies for STIM1 (Cell Signaling Technology, 5668, 1:200) or NFATc1 (Santa Cruz, sc7294, 1:200) at RT for 1 h. Cells were then washed twice with PBS, followed by incubation with 488-conjugated goat anti-mouse secondary antibody (Thermo, A32723, 1:2000) or 488-conjugated goat anti-rabbit secondary antibody (Bioss, bs-0295G, 1:500) for 1 h. Fluorescent signals were imaged by laser scanning confocal microscopy (Leica).