Rhodamine conjugated anti rabbit igg

Cells in 4-well side chambers (SPL, Pocheon, Korea) were fixed with 4% formaldehyde and rinsed with PBS. Permeabilization was performed with 0.1% Triton X-100 (Sigma) for 10 min. After being incubated in blocking solution (2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) in PBS) for 1 h, the cells were incubated with rabbit anti-UCP-1 (ThermoFisher Scientific) and p-JNK (Cell Signaling) antibodies (diluted 1:500) overnight at 4 °C. After being washed 3 times with PBS, the cells were incubated with FITC-conjugated anti-rabbit IgG (1:500, EMD Millipore) and rhodamine-conjugated anti-rabbit IgG (1:500, Vector Laboratories) secondary antibodies. DAPI (Invitrogen, Carlsbad, CA, USA) was used to stain the nuclei. Fluorescence images were captured by a confocal microscope (LSM710, Carl Zeiss Microscopy GmbH, Jena, Germany). For mitochondrial staining, MitoTracker Red (1 mM, Invitrogen) was added to the DMEM/F-12 at a concentration of 200 nM for 30 min at 37 °C. After incubation, the cells were washed with PBS, fixed with 4% formaldehyde, washed again with PBS, and immunostained.

The H9c2 cells on 4-well slide chambers (SPL, Pocheon, Korea) were fixed with 4% formaldehyde and washed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 (Sigma) for 15 min, blocked in 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h and stained with anti-NRF2 (1:200), anti-UCP-1 (1:200), anti-PPARγ (1:200), and anti-PRDM16 (1:200) antibodies at 4 °C overnight. After three rinses with PBS, the cells were incubated for 1 h with rhodamine-conjugated anti-rabbit IgG (1:500, Vector Laboratories) and FITC-conjugated anti-rabbit IgG (1:500, EMD Millipore) secondary antibodies. The nuclei were counterstained with DAPI (Invitrogen). MitoTracker Red (Invitrogen) was utilized for mitochondrial dyeing. All images were examined using a confocal microscope (LSM710, Carl Zeiss, Jena, Germany).