Radiance 2000 axiovert s100tv

Hindlimb Skm from wt and ATPIF1_H49K|^T/H mice was sliced, histologically prepared, and stained with hematoxylin/eosin by the Histology Facility at CNB‐CSIC, UAM University, Spain. Deparaffination was performed at 60°C for 1 h, followed by hydration (xylene, EtOH 100%, EtOH 90%, EtOH 70% and distilled H₂O). C₂C₁₂ cells were fixed in 4% PFA. Blockage was performed with 3% goat serum in TBS 1× and 0.5% Triton X‐100 at RT for 1 h. Dyes were incubated in 1% goat serum in TBS 1× and 0.5% Triton X‐100. Stainings were as follows: ATPIF1 (1:500), β‐F1‐ATPase (1:10,000), BODIPY 493/503 (2 μM) for LD; DAPI (1:1,000) for nuclei; and Oil Red O (0.5%) for lipid staining. Complex I activity in slices was performed as indicated for the CN in‐gel activity. Images were acquired on a Leica DMRE light microscope or by confocal microscopy using a Bio‐Rad Radiance 2000 Zeiss Axiovert S100TV. ImageJ 1.51v software was used for quantification and image analysis.

Soleus muscles were fresh frozen (OCT) or PFA-fixed, sliced, and histologically prepared (and stained with hematoxylin/eosin) by the Histology Facility at CNB-CSIC, UAM University, Spain. For freshly frozen preparations, nitrogen-chilled methylbutane was used as OCT freezing medium and slices freshly cut by cryostat. The muscle PFA-fixed sections were subjected to 20 min heat-induced epitope retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Dyes were incubated in 3% BSA, 5% horse serum in PBS with 0.1% Triton-X-100 (antibody solution). Stainings: laminin (1:1000); MFN2 (1:500); DAPI (1:1000) for nuclei. MYHs specific markers: type I fibers: MYH7, antibody clone: BA-F8 (1:50); type IIa fibers: MYH2, antibody clone: SC-71 (1:100); type IIb fibers: MYH4, antibody clone: DF-F3(1:100); type IIx fibers: antibody clone: BF-35, all but type IIx fibers (1:50). Images were acquired on a Leica DMRE light microscope or by confocal microscopy using a Bio-Rad Radiance 2000 Zeiss Axiovert S100TV.