Ec plan neofluar air

To assess the quality of the experiment and to select putative SEMs for further imaging, an overview of the majority of the SEMs was gathered using tiled scanning implemented in Zeiss LSM 700 and LSM 800 inverted confocal microscopes. SEM(s) were mounted in a 35 mm glass-bottom dish (Mattek, P35G-1.5-14-C) covered with PBS. To generate the overview images, a ×10 EC Plan Neofluar air objective (0.3 NA) or a ×20 Plan-Apochromat air objective (1.0 NA) were used with Zen software (black edition; Zeiss).

Muscle from 5-d-old pups (hindlimp) or 30-wk-old adults (soleus) was wrapped in tragacanth gum and frozen in liquid nitrogen-cooled isopentane for histological assays. Transversal 6-μm cryosections were prepared with a cryostat and samples were stored at −80°C.
For immunostainings, slides were thawed to RT and fixed in 4% paraformaldehyde for 10 min. Fixed sections were washed with PBS + 0.1% tween (PBS-T) and antigen retrieval was preformed using a low pH (20 min at 95°C and PH 6). After wash with PBS-T for 30 min, nonspecific sites blocked with 5% BSA + MOM Blocking (30 μl/ml, # VB001; R&D Systems) in PBS for 2 h. The primary antibodies (Table S1) were added O/N at 4°C in 2% BSA + 5% goat serum + 0.1% triton in PBS. Cross sections were washed three times with PBS-T and then incubated with secondary antibodies together with DAPI for 2 h. After wash again in PBS-T and dry at RT, sections were mounted in Fluoromount G (#00-4958-02; Thermo Fisher Scientific). Slides were acquired using an inverted widefield fluorescence microscope (Zeiss Cell Observer; 40x EC Plan-NeoFluar Air, NA 0.75, WD 0.71 mm).