Biotinylated ib4

The samples of epididymal white adipose tissue (eWAT) as visceral adipose tissue and subcutaneous white adipose tissue (sWAT) were fixed with 4% paraformaldehyde, and embedded in paraffin. They were sectioned into 7-mm thick slices that were stained with hematoxylin and eosin for microscopic examination. The average cell area and lipid droplets per cell were measured using the ImageJ software according to the method described in the previous study [19 ]. After deparaffinization using xylene and ethanol, immunolocalization was performed in the paraffin sections of eWAT and sWAT. Primary antibodies (4 °C, 12 h) and secondary antibodies (room temperature, 1 h) were applied to the slides upon dilution with PBS. Primary antibody used was Rabbit anti-UCP1 (Abcam, 1:250), and secondary antibodies used were Alexa 488-conjugated (Invitrogen) and HRP conjugated (DAKO). Biotinylated IB4 (Vector Labs, 1:50) was used with Alexa 488-conjugated streptavidin. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche). Images were acquired using a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). For quantification, at least ten 10 × view fields per sample were analyzed. UCP1 expression in the white adipose tissues were quantified through measuring the cumulative pixel intensity in multiple fields of view using the ImageJ software.

Tissue was immunostained as described previously (Bráz et al., 2012 (link)). Antibodies used included ATF3 (rabbit, 1:2 k, Santa Cruz Biotechnology), Fos (rabbit, 1:5 k, Oncogene), Iba1 (rabbit, 1:1 k, Wako), NeuN (mouse, 1:5 k, Sigma), β-Gal (chicken, 1:10 k, Abcam), TRPV1 (guinea pig, 1:5 k, generous gift of the David Julius lab), NF200 (mouse, 1:20 k, Sigma), CGRP (mouse, 1:10 k, Sigma), tyrosine hydroxylase (rabbit, 1:5 k, Millipore), and biotinylated IB4 (goat, 1:500, Vector Labs). Fluorescent secondary antibodies were used at a 1:1 k dilution; streptavidin-conjugated fluorophore was used at 1:5 k.