A new therapeutic target for the prevention of heartfailure due to aorticstenosis
Snap frozen hearts were crushed and200-400 mg of tissue was mixed with 50 mM Tris–HCl . The samples were homogenized using an ultrasonic cell disruptor keeping the sample on ice and they were filtered using nylon mesh. Next, the samples were centrifuged at 4 °C for 15 min at 1000 g.
For immunofluorescence, hearts were fixed in 4% formaldehyde, dehydrated through 15% sucrose in PBS and then 30% sucrose overnight at 4 °C, and embedded in Tissue-Tek® OCT compound . Cryostat sections were blocked and permeabilized for 1 h at RT in PBS containing 0.3% Triton X-100 , 5% BSA , and 5% normal goat serum . Sections were then incubated overnight at 4 °C with anti-GFP diluted in PBS containing 0.3% Triton X-100 and 2.5% normal goat serum.
For cell immunofluorescence, adult mouse ventricular myocytes and neonatal rat ventricular myocytes were fixed with 4% paraformaldehyde in PBS for 10 min. Cells were then washed 1–3 times with PBS and blocked with 2% BSA for 1 h at RT. Samples were incubated overnight at 4 °C with primary antibodies . After 1–3 washes with PBS, cells were incubated with Alexa Fluor secondary antibodies for 1 h at RT.
Cells were harvested 3 days after transfection, lysed, frozen and thawed three times, and digested with benzonase . The final supernatant containing the virus was then purified on an iodixanol gradient in an optiseal polypropylene tube . The concentrated and purified viral fraction was collected between the 40% and 60% iodixanol layers after ultracentrifugation .
Adult mice were anesthetized and maintained on 1–2% isoflurane. A skin incision was made on the medial face of the hindlimb, and the femoral vein was exposed. A dose of 3 × 10viral genomes /mouse in 50 µl saline was injected using a 31G insulin syringe, and the skin was closed with a 6/0 silk thread. AAV-9 was used for in vivo delivery.To verify correct viral transfection, 875 µg of D-luciferin was administered to mice in a volume of 50 µl by intraperitoneal injection.
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