Rabbit monoclonal anti mfn2

Fresh heart tissues were fixed in 4% paraformaldehyde, transferred to 18–10% sucrose in PBS, and embedded in OCT. Samples were sectioned at 8 μm, air-dried and stored at −80°C until used. Frozen slides were then thawed, permeabilized, blocked with 3% donkey serum (Jackson ImmunoResearch catalog number 017-000-121) in PBS, and subjected to staining by a rat monoclonal anti-Lamp1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number sc-1992; 1:300) or a rabbit monoclonal anti-Mfn2 (Cell Signaling, Danvers, MA, catalog number 9482; 1:100 at room temperature, 1 h). Upon completion of secondary antibody incubation with Alexa Fluor 448 conjugated donkey anti-rat IgG (Thermo Scientific; catalog number A-21208; 1:300) or Alexa Fluor 647 conjugated donkey anti-rabbit IgG (Thermo Scientific; catalog number A-31573; 1:300), the slides were washed, sealed with DAPI/antifade mounting solution (Thermo Fisher Scientific, Rockford, IL; catalog number 36931), and examined. Images were acquired with a LSM 510 confocal microscope equipped with an Axio Observer Z1 motorized inverted microscope and Zen software (Carl Zeiss Microscopy) and analyzed offline with Imaris software (version 9.5, Bitplane).

Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin-streptomycin solution, 0.25% trypsin/EDTA, and phosphate-buffered saline were purchased from Gibco. Fetal bovine serum (FBS) was purchased from Biological Industries. Rabbit monoclonal anti-Mfn1(Cat#: 14739S), rabbit monoclonal anti-Mfn2(Cat#: 9482S), rabbit monoclonal anti-Opa1(Cat#: 80471S), rabbit polyclonal anti-Drp1(Phospho Ser616 Cat#: 4494S), rabbit monoclonal anti-AMPK_α (Phospho Thr172 Cat#: 2535S), and rabbit monoclonal anti-β-actin(Cat#: 4970T) were purchased from Cell Signaling Technology. Rabbit monoclonal anti-Fis1(Cat#: ab156865) and rabbit monoclonal anti-DRP1(Cat#: ab184247) were purchased from Abcam. DMSO, Mito-Tracker, BCA Protein Quantification Kit, RIPA lysis buffer, protease inhibitor cocktail, phosphatase inhibitors, SDS-PAGE Gel Preparation Kit, and other Western blotting reagents were purchased from Yeasen (Shanghai, China). Annexin V-Alexa Fluor 647/PI Apoptosis Detection Kit was purchased from Fcmacs (Nanjing, China). The mitochondrial membrane potential assay kit with JC-1 was purchased from Beyotime (Nanjing, China). Unless otherwise indicated, all other chemicals and reagents were purchased from Sigma-Aldrich.