Mouse anti coxiv

Primary MN were grown on 13 mm glass cover slides, and then fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. Cover slides were then washed in PBS and permeabilized with 0.5% Triton in blocking solution containing 5% donkey serum (Jackson Laboratories) and 1 mg/mL BSA (Sigma) for 5–30 min. After three washes, cells were incubated with appropriate antibodies overnight at 4°C in blocking solution [rabbit anti-Dicer (Abcam,1:100), rabbit anti-Ago2 (Abcam,1:200), chicken anti-NFH (Abcam and Millipore, 1:1000), mouse anti-neurofilament phosphorylated (SMI31)/non-phosphorylated (SMI32) (Covance, 1:500), mouse anti-Rab7 (Abcam, 1:300), mouse anti-TrkB (BD Bioscience, 1:200), mouse anti-COX IV (1:200; Cell Signaling Technology)]. After having been washed, secondary antibodies (2 μg/mL Jackson Laboratories, ThermoFisher) in blocking solution were added for 2 h at room temperature. Phalloidin was used to visualize F-actin, and added when necessary to the secondary antibody. For axonal visualization, cells were incubated for 45 min with Alexa Fluor 594/405-conjugated phalloidin (1x, Abcam), diluted in PBS, then washed and mounted with ProLong Gold (Life Technologies). For mitochondria staining, the cells were incubated with Mitotracker Deep Red FM (100 nM, Thermo Fisher) for 30 min at 37°C, and then washed 3 times with PBS prior to fixation.

Antibodies and reagents were purchased from: mouse anti-flag (F1804, Sigma, USA); rabbit anti-flag (ab205606, Abcam, USA); rabbit anti-AFG3L2 (14631-1-AP, Proteintech, USA); rabbit anti-ubiquitin (ET-1609-21, HUABIO, China); mouse anti-COXIV (11967S, Cell Signaling Technology, USA); rabbit anti-vimentin (5741, Cell Signaling Technology, USA); rabbit anti-GAPDH (10494-1-AP, Proteintech, USA); anti-flag magnetic beads (HY-K0207, MCE, USA); cycloheximide (CHX, 66-81-9, Selleck, USA); MG132 (HY-13259, MCE, USA); chloroquine (CQ, HY-17589A, MCE, USA).

Immunostaining was performed as described previously (Cheng et al., 2017 (link)), with the following primary antibodies: mouse anti‐cardiac troponin T (MS‐295‐P, Thermo Scientific, 1:100), mouse anti‐COX IV (#11967, Cell Signaling, 1:100), or rabbit anti‐Drp1 (#8570, Cell Signaling, 1:100). Cardiomyocyte cell surface area was measured using ImageJ. Manders’ coefficient for evaluation of colocalization was analyzed using the ImageJ plugin Coloc 2.