Hoechst 3342

Immunostaining was performed using a standard protocol. After stimulation with BMP-2 for 7 days, hBMSCs were incubated with primary rabbit polyclonal antibody against COL1A1 (Abcam, USA) and mouse polyclonal antibodies against VEGF (Abcam, USA) or mouse polyclonal antibodies against COL1A1 (Abcam, USA) overnight at 4 °C, and then primary antibodies were detected using Cy3- or FITC-conjugated anti-mouse or anti-rabbit IgG secondary antibody (Life Technologies, USA). After the final wash, nuclei were counterstained with Hoechst 3342 (Life Technologies, USA) in PBS for 5 min before imaging. The stained sections were immediately observed by laser confocal microscopy (Zeiss, Germany).

For the microscopic analysis of autofluorescence, cells were grown on coverslips for 16hr, treated with 50 μM for 3 h before being fixed with 4% PFA. After mounting with Fluoromont-G mounting medium (Southern biotech), coverslips were observed with a Zeiss confocal microscope and fluorescence spectra induced by menadione treatment was evaluated in multiple regions of interest (ROI) within the same cell.
For indirect immunofluorescence analysis, cells were fixed on coverslips for 15 min with 4% PFA and then permeabilized for 5 min with Triton 0.2% at room temperature. AIF protein was detected by incubation with an anti-AIF pAB (1:100 dilution in PBS / 1% BSA / 2% goat serum) followed by Alexafluor-647-conjugated goat anti-rabbit (Life Technologies). DNA was stained with Hoechst 3342 or TO-PRO-3 (Life Technologies). Sample slides were then mounted using the reagent Fluoromont-G (Southern Biotech) and observed by confocal microscopy (LSM-510; Carl Zeiss Microimaging) equipped with an X63 objective.

To visualize nascent t‐tubule formation, we used di‐8‐ANEPPS (Life technologies), a lipophilic voltage‐sensitive dye that localizes to t‐tubules in mature hPSC‐CMs. As described previous, we prepared 100 µmol/L di‐8‐ANEPPS in 20% (w/v) Pluronic‐F127 (Sigma) in DMSO from a stock solution of 2 mmol/L di‐8‐ANEPPS in DMSO and added enough cell‐culture medium to incubate cells in 10 µmol/L di‐8‐ANEPPS for 15 minutes at 37°C. The medium was changed, and cells were imaged 30 minutes later. Mitochondria in live cells were labelled with MitoTracker Red (Life Technologies) at a concentration of 50 nmol/L for 10 minutes at 37°C. The cells were washed once with warm PBS, and new warm culture medium was added immediately before imaging. Nuclei were visualized by incubating live cells in medium with 1 µg/mL Hoechst 3342 (Life Technologies) for 5 minutes at 37°C, and fresh medium was added. A confocal microscope (Leica TCS SP5) was used to image t‐tubules labelled with di‐8‐ANEPPS and mitochondrial labelled with MitoTracker Red.

Selected LCLs were obtained from patients with MD according to the genotype. LCLs undergoing TWEAK treatment (250 ng/mL) for 48 h were fixed using fresh methanol: DMSO (4:1) and stored at −20°C until used. LCLs were then rehydrated, blocked, stained, and mounted as previously described (34 (link)). Primary antibodies were used as follows: a mouse monoclonal antibody against Fn14 (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-56250, 1:50) and a rabbit polyclonal antibody against NF-κB p105/p50 (Abcam, Cambridge, UK; #ab7971, 1:50). As secondary antibodies, we used Alexa-555-conjugated goat anti-mouse (Life Technologies, Carlsbad, CA, USA; #A-21422, 1:500) and Alexa-633-conjugated goat anti-rabbit (Life Technologies, Carlsbad, CA, USA; #A-21071, 1:500), respectively. For nuclei staining, we used Hoechst 3342 (Life Technologies, Carlsbad, CA, USA; #H1399, 1:1,000). A laser scanning confocal microscope LSM 710 (Carl Zeiss, Oberköchen, Germany) was used for image collection and the Zeiss browser software program ZEN black edition was used to acquire and export the data. All images were taken with the same laser intensity settings on the microscope and final image processing and labeling were performed with ImageJ.