Laser scan microscope lsm 700

Induced myogenic cells (iMCs) grown on Matrigel-coated 8-well IbiTreat slides (Ibidi) were fixed with 3.7% Formaldehyde (10 min). Cells were permeabilized with 0.1% Tween 20 in TBS and blocked in 0.1% Triton X-100 + 1% FBS in TBS for 30 min. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C on a shaker (Desmin (1:1000, Abcam ab15200), fast MyHC (1:200, Sigma-Aldrich M4276), MyHC3 (1:200, Santa-Cruz sc-2064), Myogenin (1:800, Abcam ab1835), PAX7 (1:200, Santa-Cruz sc-81648), TUJ1 (1:1000, Sigma-Aldrich T8578)). Next day, secondary antibodies were also diluted in blocking solution and again incubated overnight at 4 °C on a shaker (AlexaFluor 568 goat anti-mouse (1:1000, Thermo Fisher A11031), AlexaFluor 568 donkey anti-rabbit (1:1000, Thermo Fisher A10042), AlexaFluor 488 goat anti-mouse (1:1000, Thermo Fisher A11001)). Hoechst (1:10,000 in DPBS) was incubated for 5 min at RT. Confocal immunofluorescence imaging was performed using the Laser Scan Microscope LSM 700 (Carl Zeiss, Jena, Germany) and for mosaic image acquisition a Leica DMI 6000 B microscope equipped with a XY scanning stage (Leica Microsystems) was used.

Sections were thawed and dried from −20 °C to RT for 45 min and then fixed for 5 min in −20 °C cold acetone. Sections were dried again for 10 min at RT and then blocked with 5% BSA + 3% goat serum in DPBS (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at RT in a humidity chamber. Sections were washed once with DPBS and then incubated with the primary antibody in 1% BSA in DPBS for 2 h at RT in a humidity chamber (human Spectrin (1:100, Novocastra NCL-SPEC1), human Lamin A/C (1:4000, Abcam ab108595)). Secondary antibodies were diluted in DPBS and incubated for 45 min at RT in a humidity chamber (Alexa Fluor 568 goat anti-rabbit (1:500, Thermo Fisher A11036), Alexa Fluor 647 goat anti-mouse (1:500, Thermo Fisher A21236)). Hoechst (1:5000 in DPBS) was incubated for 5 min at RT. Sections were mounted on microscope slides using Aqua-Poly/Mount (Polysciences, Inc.) and were left to dry overnight at 4 °C. Confocal immunofluorescence imaging was performed using the Laser Scan Microscope LSM 700 (Carl Zeiss).