Abs137082

The treated-side SS (n = 5 per group) cryosection slides were fixed in 4% paraformaldehyde for 30 minutes, rinsed in PBS, placed in 0.1 M glycine (diluted in PBS; Fisher Scientific) for 30 minutes, and washed again in PBS. They were then covered with blocking solution (0.2% Triton X-100; 2% bovine serum albumin in PBS) for 1 hour at room temperature. Primary antibodies against UCP1 (an indicator of BAT activation or white fat browning) (diluted 1:100; NB100-2828; Novus Biologicals), tyrosine hydroxylase (TH) (an indicator of activation of sympathetic nerve fibers) (diluted 1:500; 66334-1-Ig; Proteintech), laminin (diluted 1:500; L9393; Sigma-Aldrich), and perilipin (diluted 1:500; abs137082; Absin) were diluted in a block mix and added to the sections for overnight incubation at 4°C. After a PBS rinse, the sections were incubated with a mixture containing FITC-conjugated (diluted 1:500; SA00003-8, SA00003-2; Proteintech) and Cy3-conjugated (diluted 1:500; SA00009-3, SA00009-1; Proteintech) secondary antibodies at room temperature for 120 minutes. After a PBS rinse for 2 × 10 minutes, the slides were covered with DAPI containing antifade mounting medium.

Samples were fixed in 4% paraformaldehyde for 30 minutes, rinsed in PBS, placed in 0.1 M glycine (Thermo Fisher Scientific, diluted in PBS) for 30 minutes, and washed again in PBS. They were then covered with blocking solution (0.2% Triton X-100, 2% bovine serum albumin in PBS) for one hour at room temperature. Primary antibodies against UCP1 (indicator of activation of beige adipose tissue or white fat browning) (NB100-2828, Novus Biologicals, USA, diluted 1:100), tyrosine hydroxylase (indicator of activation of sympathetic nerve fibres) (66334-1-Ig, Proteintech, USA, diluted 1:500), laminin (L9393, Sigma-Aldrich, diluted 1:500), and perilipin (abs137082, Absin (China), diluted 1:500) were diluted in a blockmix and added to the sections for overnight incubation at 4°C. After a PBS rinse, the sections were incubated with a mixture containing fluorescein isothiocyanate-conjugated (SA00003-8, SA00003-2, Proteintech, diluted 1:500) and Cy3-conjugated (SA00009-3, SA00009-1, Proteintech, diluted 1:500) secondary antibodies at room temperature for 120 minutes. After a PBS rinse for ten minutes (done twice), the slides were covered with DAPI containing antifade mounting medium.