Anti prdm16 antibody

Adipose tissues were fixed in 4% formaldehyde overnight. After paraffin embedding and sectioning at 5 µm, the sections were stained with hematoxylin and eosin (Beyotime) according to standard protocols. For immunohistochemistry, the sections were blocked with 5% BSA in PBS for 1 h, and incubated with anti‐PRDM16 antibody (R&D systems) at 4 °C overnight. The results were imaged with a light microscopy Motic BA600 (Motic Germany GmbH) and a slice scanner Pannoramic MIDI II (3DHISTECH). PRDM16‐Luc SVCs were differentiated into adipocytes and treated with DHT (1 µm, Meilunbio) or DMSO for 24 h. The adipocytes were washed with PBS, fixed in 4% formaldehyde for 10 min, permeabilized in PBST (PBS +0.1% TritonX‐100) for 10 min, blocked in 5% BSA for 30 min on ice, and incubated with Anti‐UCP1 antibody (Abcam) at 4 °C overnight. The 2nd day, after washing for three times, the adipocytes were incubated with fluorescently labeled goat anti‐rabbit IgG (Thermo Fisher Scientific) for 1 h, followed by incubation with BODIPY 493/503 (Thermo Fisher Scientific) plus Hoechst 33342 (Merck) for 20 min. Laser scanning microscopy 800 (ZEISS) with oil‐immersion objective lens was used for confocal imaging.

FBS (cat# 16000-044), TRIzol reagent (cat# 15596-018), DMEM/F-12
(cat# 11330-032), and DMEM (cat# 11965-092) were purchased from
Invitrogen (Carlsbad, CA, USA). SYBR-Green fluorescent dye (cat# 4368577)
and TaqMan miRNA probes were purchased from Applied Biosystems (Foster City, CA,
USA). Collagenase type II (cat# c6885), oligomycin (cat# 75351), FCCP
(cat# C2920), rotenone (cat# R8875), indomethacin (cat# I-7378),
dexamethasone (cat# D-1756), isobutylmethylxanthine (cat# I-5879),
rosiglitazone (cat# R-2408), T3 (cat# T-2877) and the MystiCq microRNA
qPCR Assay (cat# MIRRM02) were purchased from Sigma (Deisenhogfen,
Germany).
For western blotting, anti-UCP1 antibody (cat# 14670) was purchased from Cell
Signaling Technologies (Danvers, MA, USA), anti-PRDM16 antibody (cat#
AF6295) was purchased from R&D Systems (Tustin, CA, USA)21 (link),
the anti-GAPDH antibody (cat# sc-25778) was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA)38 (link). For immunohistochemistry,
anti-UCP1 antibody (cat# ab10983) was purchased from Abcam (Cambridge, MA,
USA)39 (link). The antibodies used for flow cytometry, including
anti-CD45 (cat# 103121), CD11b (cat# 101207), and F4/80 (cat#
123115)26 (link), were purchased from BioLegend (San Diego, CA,
USA).