Tecnai biotwin spirit

Control adult skin fibroblasts, control neonatal skin fibroblasts or skin fibroblasts obtained from patients 1 and 2 were cultured on Aclar coverslips, fixed with 1.25% paraformaldehyde, 2.5% glutaraldehyde, 0.03% picric acid followed by osmication and uranyl acetate staining, dehydration in alcohols and embedded in Taab 812 Resin (Marivac Ltd, Nova Scotia, Canada). Sections were cut with Leica ultracut microtome, picked up on formvar/carbon coated copper slot grids, stained with 0.2% Lead Citrate, and imaged under the Phillips Tecnai BioTwin Spirit transmission electron microscope. Muscle electron microscopy studies were performed as previously described (Malfatti et al., 2014 (link)).

HeLA cells transfected with control siRNA or 2 different siRNAs targeting QIL1 and HCT116 stable cell lines expressing FF2 shRNA or 3 independent shRNAs targeting QIL1 were grown to 60% confluency in 6-cm culture dishes and fixed with 1.25% paraformaldehyde, 2.5% glutaraldehyde, 0.03% picric acid followed by osmication and uranyl acetate staining, dehydration in alcohols and embedded in Taab 812 Resin (Marivac Ltd, Nova Scotia, Canada). Sections were cut with Leica ultracut microtome, picked up on formvar/carbon-coated copper slot grids, stained with 0.2% Lead Citrate, and imaged under the Phillips Tecnai BioTwin Spirit transmission electron microscope. For the in vivo analysis, Drosophila muscle tissues from third instar larvae were dissected in Ca²⁺ free buffer, fixed overnight at 4°C and processed as described above.