Horseradish peroxidase conjugated anti rabbit sc 2004

Tissue samples were homogenized by sonication in RIPA buffer at 4 °C supplemented with protease and phosphatase inhibitors cocktail. Tissues extracts were centrifuged at 12,000 rpm at 4 °C for 20 min to remove insoluble material. After quantification, 20 µg of total protein was subjected to polyacrylamide gel electrophoresis (SDS PAGE 12%) and transferred to a polyvinylidene fluoride membranes (PVDF). Then, membranes were blocked 1 h in 5% BSA-TBST, 20 mM Tris-base, pH 7.6, 137 mM NaCl and 0.1% Tween 20 and incubated with the respective primary antibody β-ACTIN (bs-0061R; lot#AH01032107, Bioss antibodies, Woburn, MA, USA, 1/4000) and anti-UCP-1 (ab-10983, lot#GR214286-5; Abcam, Cambridge, UK, 1/3500) overnight at 4 °C. After washing, membranes were incubated with Horseradish Peroxidase conjugated anti-rabbit (sc-2004, lot#22905; Santa Cruz, Dallas, TX, USA, 1/10,000) as secondary antibody for 2 h at room temperature with constant shaking. The protein signals were detected using ECL and autoradiography films (Carestream Medical X-ray Green/MXG film). Images were analysed with Fiji software 1.54f.