Ma3 920

Proteins in lysates of 2-3month old wildtype and Ehd1-heterozygous quadriceps muscles, (n ≥ 2), were transferred to membranes and were immunoblotted with anti-BIN1 (1:1000, sc-23918, Santa Cruz, Dallas, TX), rabbit polyclonal anti-EHD1 (1:5000, [10 (link)]), anti-DHPR (1:1000, ma3-920, Thermo Scientific, Rockford, IL), anti-Junctophilin 2 (1:1000, sc-51313, Santa Cruz, Dallas, TX), anti-Junctophilin 1 (1:1000, 40–5100, Thermo Scientific, Rockford, IL) antibodies. Secondary antibodies, goat anti-rabbit, and goat anti-mouse conjugated to horseradish peroxidase (Jackson ImmunoResearch) were used at a dilution of 1:2500. Blocking and antibody incubations were done in Starting Block T20 (Invitrogen, Grand Island, NY) for all antibodies and rinsed with TBS-T. Memcode and gelcode stains were used to mark proteins for loading controls (Life Technologies, Grand Island, NY). Chemiluminescence substrate, Kodak Biomax MS film, and a UVP BioSpectrum Imaging System (Upland, CA) were used for detection.

Mutant and WT cultures on glass coverslips were fixed with 4% PFA solution for 15 min and rinsed 3× with 1× PBS solution. Cells were then blocked with donkey serum blocking buffer (2.5 mL Donkey Serum + 2.5 mL BSA + 40 mL sterile water + 5mLs PBS 10x) for 1 h after a 15-min permeabilization period with 0.1% triton in PBS. Primary antibodies for MyoD, Pax7, desmin (Thermofisher Scientific PA5-16705, MyHC DSHB, A4.1025-s), and Ryanodine receptor (RyR) (Millipore, AB9078), dihydropyridine receptor (DHPR) (Thermofisher Scientific,MA3920) were added to blocked cultures and incubated overnight at 4 °C. The secondary antibodies donkey anti-mouse and anti-rabbit (Thermofisher Scientific, A10037, A-21206) were added the next day at a 1:250 dilution. Coverslips were rinsed 3× in 1× PBS mounted on glass slides with Prolong gold mounting medium with DAPI (Life Technologies P36931) after a 2-h incubation period for imaging. Images were taken with Axioskop 2 mot plus upright spinning disk confocal microscope (Carl Zeiss) that had been connected to a XCite 120 Fluorescence Illumination system (EXFO) beam with multi-spectral laser scanning software, Volocity version 6.3.0 (Perkin Elmer).

Transverse tissue sections (10 µm thickness) were cut from the middle part of the lateral head of the gastrocnemius and soleus muscles using a cryostat (CM1950; Leica, Wetzlar, Germany) at −25 °C and mounted on amino silane-coated glass slides. Subsequently, the transverse sections (10 µm thickness) were fixed in 4% paraformaldehyde and rinsed with phosphate-buffered saline (PBS; pH 7.4). The sections were incubated in PBS containing 1% normal goat serum and 0.3% Triton X-100 at 4 °C for 1 h. The sections were then incubated with mouse monoclonal anti-DHPRα1 antibodies (MA3-920; Thermo Fisher Scientific, Waltham, MA, USA; 1:100) and rabbit polyclonal anti- SERCA1 antibodies (22361-1-AP; Proteintech, Rosemont, IL, USA; 1:500) in PBS containing 0.3% Triton X-100 at 4 °C for 24 h. Subsequently, the sections were incubated with anti-mouse Alexa Fluor 488 Conjugate (A11001, Thermo Fisher Scientific, Waltham, MA, USA) and anti-rabbit Alexa Fluor 555 Conjugate (4413S, Cell Signaling Technology, Danvers, MA, USA) secondary antibodies diluted at 1:1000 in PBS for 1 h at 25 °C. After rinsing with PBS, the sections were embedded in DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Thereafter, the stained sections were observed under a microscope (BZ-X800; Keyence, Osaka, Japan).