Ab184032

The tissue was fixed with 4% paraformaldehyde, embedded in paraffin and sectioned. The paraffin-embedded sections were dewaxed and rehydrated, and EDTA was used for antigen repair. At room temperature, sections were incubated with 3% H₂O₂ to quench endogenous peroxidase activity. Afterwards, samples were blocked with BSA and incubated overnight at 4℃ with specific primary antibodies, including anti-CD147 (ab212057, 1:200, Abcam, Cambridge, USA), anti-ACOX1 (ab184032, 1:200, Abcam), anti-FASN (ab128856, 1:200, Abcam). Later, samples were incubated for 1 h at 37°C with HRP-labeled goat anti-Rabbit secondary antibodies. The samples were then dyed with DAB and hematoxylin before sealing.

Immunoprecipitation was performed to identify mtHSP70 acetylation sites, SIRT3–mtHSP70 interaction, and mtHSP70–ACOX1 interaction according to our previous study [35 (link)]. Briefly, in order to conduct the immunoprecipitation analysis, we lysed the cells in NP-40 buffer (Beyotime, Nantong, China, p0013F) with the addition of a protease inhibitor cocktail (Beyotime, Nantong, China, P1010). The cell lysate was then mixed with anti-SIRT3 (ab217319; abcam, Cambridge, UK), anti-mtHSP70 (ab2799; abcam, Cambridge, UK), anti-ACOX1 (ab184032; abcam, Cambridge, UK), anti-FLAG-tag (ab1162; abcam, Cambridge, UK), or anti-HA tag (ab18181; abcam, Cambridge, UK) at 4 °C overnight, followed by the addition of protein A/G beads (P2012; Beyotime, Nantong, China). NP-40 buffer was used to wash the immunocomplexes. Finally, the Western blot analysis was performed. Additionally, due to the hepatocytes of yellow catfish not being satisfied for the immunoprecipitation experiment, all this immunoprecipitation was conducted by using another cell line (HepG2 cell line), similar to our previous study [35 (link)].
To identify the protein levels of SIRT3, mtHSP70, ACOX1, TIMM44, GAPDH, Histone H3, Ub LONP1, and acetyl lysine, a Western blot analysis was performed according to our previous study [35 (link)]. Among these antibodies, acetyl lysine was used to test mtHSP70 acetylation after immunoprecipitation and also the immunofluorescence staining. In brief, the protein was loaded onto the SDS−PAGE gel and then transferred to the PVDF membrane. Membranes were blocked with 5% skimmed milk and then incubated with the following primary antibodies overnight at 4 °C. Membranes were then incubated with corresponding secondary antibodies: HRP-conjugated anti-rabbit IgG antibody (7074; CST), HRP-conjugated mouse anti-rabbit IgG (5127; CST), or HRP-conjugated anti-rabbit IgG antibody (light chain-specific) (93702; CST). After further washing, membranes were visualized via ECL (1705060; Bio-Rad, Hercules, CA, USA.). These antibodies from other various species, including human, rat, and mouse, were used in our previous study. Studies have shown that these antibodies are feasible for yellow catfish [22 (link),24 (link),35 (link)].

Immunohistochemistry staining was performed. Briefly, the sections were preincubated with 3% hydrogen peroxide and blocked with 2.5% normal horse serum. Then, the slides were incubated with anti-ACOX1 (ab184032, 1:200, Abcam, Cambridge, United Kingdom), PPARα (ab233078, 1:200, Abcam, Cambridge, United Kingdom), SCD (ab19862, 1:300, Abcam, Cambridge, United Kingdom), and FASN (ab22759, 1:200, Abcam, Cambridge, United Kingdom) primary antibodies overnight at 4°C and then with the secondary antibody (GB25301, 1:500, Servicebio Co., Ltd., Wuhan, China) at room temperature for 60 min. ImageJ 1.4 software (NIH, Bethesda, MA) was used to quantify the protein expression.