For ORO staining, tibialis anterior muscles were sectioned (10 μm) and immediately fixed in ice cold formaldehyde (3.7%; Thermo Fisher Scientific) for 1 h. Sections were serially washed in Milli-Q water (MilliporeSigma) and stained in ORO working solution prepared as previously described (Huot et al., 2020a (link)); for 45 min at room temperature in the dark. Following ORO staining, sections were again serially washed in Milli-Q water and then rinsed in running tap water for 10 min. Sections were mounted in 50% glycerol in PBS and photographed using an Axio Observer.Z1 motorized microscope (Zeiss). Entire ORO-stained sections were analyzed for signaling intensity and area of positive staining using ImageJ software.
Axio observer z1 motorized microscope
Manufactured by Zeiss
Sourced in Germany
The Axio Observer.Z1 is a motorized microscope designed and manufactured by Zeiss. It features a motorized focus drive, stage, and condenser. The microscope is capable of various contrast techniques, including brightfield, darkfield, DIC, and fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Milli q water, by Merck Group (1 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Trap kit, by Merck Group (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Alexa fluor594 labeled, by Thermo Fisher Scientific (1 mentions)
Polyclonal anti laminin, by Merck Group (1 mentions)
4 protocols using axio observer z1 motorized microscope
1
Muscle Lipid Quantification via ORO
2
Osteoclast Differentiation Assay
3
Quantifying Differentiated Myotube Size
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To assess size, differentiated C2C12 myotubes were fixed in ice-cold acetone:methanol for ten minutes, blocked for 1 h at room temperature and incubated overnight at 4°C with an anti-myosin heavy chain antibody (MF-20, 1:100, Developmental Studies Hybridoma Bank). The following day, myotubes were incubated for 1 h at room temperature with an AlexaFluor 594-labeled secondary antibody (A11032, 1:500; Invitrogen). Myotubes were observed under an Axio Observer.Z1 motorized microscope (Zeiss) and analysis was performed by measuring the diameter of the narrowest portion along the multi-nucleate fibers (n=400 fibers per condition) using ImageJ software (Schneider et al., 2012 (link)). Three biological replicates (n=3) were performed for each experimental condition.
4
Muscle Fiber Cross-sectional Area Analysis
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Ten‐micrometre‐thick cryosections of tibialis anterior muscles taken at the mid‐belly were incubated overnight with polyclonal anti‐laminin (1:1000; Sigma‐Aldrich) to label the basement membranes of the muscle cells.20 Sections were then incubated for 1 h with Alexa Fluor 594‐conjugated secondary goat anti‐rabbit IgG (Invitrogen, Carlsbad, CA, USA). All samples were observed under an Axio Observer.Z1 motorized microscope (Zeiss, Oberchoken, Germany), and images were recorded for morphometric examination. For determination of the cross‐sectional area, muscle fibre size (n = 300–500 per sample) was measured by using the ImageJ 1.43 software.
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