Anti ucp1 antibody

Fresh BAT, epididymal WAT, liver, and qM tissues were fixed in 10% (v/v) formalin, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin (H&E) or immunohistochemically using anti-F4/80 antibody (#70076; Cell Signaling Technology, Danvers, USA) and anti-UCP1 antibody (sc-518024, Santa Cruz Biotechnology, Santa Cruz, USA)
[24] (link). In brief, the sections were heated (65°C, 2 h), dewaxed, rehydrated, and boiled (2 min) in sodium citrate (10 mM, pH6.0) for antigen retrieval. The slides were then treated with 3% H
₂O
₂ for 30 min to eliminate endogenous peroxidase activity and blocked with 5% BSA for 1 h. The slides were incubated with primary antibody at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (A0208; Beyotime, Shanghai, China) for 1 h at room temperature. Color development was performed by incubation with DAB reagent (P0203; Beyotime), and the slides were counterstained with hematoxylin according to manufacturer’s protocols.

Cells grown on poly-L-lysine-pretreated coverslips were fixed with 4% p-formaldehyde followed by washing with phosphate-buffered saline (PBS) and then subjected to permeabilization with 0.25% Triton X100 (Sigma-Aldrich, MO, USA). Cells were washed with PBS three times, blocked with 1% Bovine Serum Albumin (BSA) in PBS-T for 1 h, and incubated with polyclonal anti-UCP1 antibody (1:200 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, followed by three washes with PBS. Cells were then incubated with fluoresceine isothiocyanate (FITC)-conjugated antigoat secondary antibody (1:400 dilutions). 4,6-diamino-2-phenyl indole (DAPI) (Thermo Fisher Scientific, Boston, MA, USA) was used to stain nuclei of cells. Florescence images were captured using a confocal laser scanning microscope LSM700 (Carl Zeiss, Oberkochen, Germany). Analysis of images (control and curcumin-treated) was performed by software Zen 2009 (Carl Zeiss, Germany). For staining of mitochondria, Mito Tracker Red (1 mM, Cell Signaling Technology, Beverly, MA) was directly added to the growing media at a concentration of 20–25 nM, and cells were kept for 30–40 min at 37°C. After incubation, cells were fixed in 4% p-formaldehyde, followed by a single wash with PBS and immunostaining (4 (link)).

Protein expression measurements were conducted using cell lysates from maturing brown adipocytes at Day 0, Day 4, Day 6 (with and without isoproterenol treatment). Cell lysates were prepared in 1.2X Laemelli buffer, sonicated at high speed, 30 s ON/OFF cycles, for 10 minutes and denatured at 95 °C for 5 minutes. Standard SDS–PAGE was performed using 10–12% resolving gels and transferred to a nitrocellulose membrane for probing. The following proteins were probed (1) UCP1 (using Abcam anti-UCP1 antibody) (2) PPARγ (using Santa Cruz anti-PPARγ antibody) (3) α-TUBULIN (using Sigma anti- α-TUBULIN antibody).