Ab184032

The distribution of SIRT3 into the mitochondrion (MT), the acetylation level, and the co-localization of mtHSP70 and ACOX1 were analyzed through immunofluorescent staining, based on a study by Jeong et al. [30 (link)]. Generally, samples were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 min. The samples were blocked for 30 min in PBST with 1% BSA and 22.52 mg/mL of glycine, followed by incubation with anti-SIRT3 (ab217319; abcam, Cambridge, UK), anti-acetyl Lysine (ab21623; abcam, Cambridge, UK), anti-mtHSP70 (ab2799; abcam, Cambridge, UK), or anti-ACOX1 (ab184032; abcam, Cambridge, UK) overnight at 4 °C. Our previous studies have proved that antibodies from other various species can be used on yellow catfish [2 (link)]. Then, incubation with a goat anti-rabbit IgG H&L secondary antibody (ab150079; abcam, Cambridge, UK) continued for 1 h at room temperature. The nucleus and mitochondrion were stained with DAPI and MitoTracker Red CMXRos, respectively. Fluorescent microscopy and the laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) were used for the observation.

Ten-micron thick sections of formalin-fixed paraffin-embedded kidney were used for haematoxylin and eosin staining, while five-micron thick sections were used for immunohistochemical staining. Scanned haematoxylin and eosin-stained sections were used to measure glomerular area in QuPath (36 (link)), from which glomerular volume was calculated using the Weibel and Gomez formula (37 (link)). Thirty glomerular tufts per sample were manually annotated at random throughout the renal cortex, from a minimum of six animals per experimental group. Kidney sections were stained with anti-ACOX1 antibody (ab184032, Abcam) using the VECTASTAIN Elite ABC-HRP peroxidase kit (PK-6200, Vector Laboratories). An IgG isotype control (ADI-950-231-0025, Enzo Life Sciences) and no antibody control were used to confirm the specificity of staining. All slides were digitized using an Aperio AT2 Digital Slide Scanner (Leica Biosystems).

The left part of isolated rat BAT was used for Western blotting. Eight samples from each experimental group were pooled by three + three + two to obtain three samples per group. Thereafter, protein content was estimated [29 (link)]. Primary antibodies were used against ACOX1 (1 : 1000, ab-184032; Abcam, Cambridge, UK), β-actin (1 : 2000, ab-8226; Abcam, Cambridge, UK) and ACOX3 (1 : 500, sc-373977; Santa Cruz Biotechnology, Texas, USA). Immunoreactive bands in Western blots were quantified using ImageJ software (NIH, Bethesda, USA). The normalized target protein was averaged from three independent experiments and two representative bands per group were shown. The average value obtained in the euthyroid group was taken as 100%. Those from hypothyroid groups were expressed as percentages of the euthyroid group.