Protocol for measuring force in skinned diaphragm muscle fibers of myopathic SEPN1 knockout mice following chronic tauroursodeoxycholic acid treatment

Figures

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  Figure 1. Stereo microscope setup and T-clip making process (A) Stereo microscope ZEISS Discovery. V8 equipped with 1× lens (higher magnification) or 0.63× lens (larger field and larger working distance), camera Axiocam 208 color mounted on an Intermediate Photo Tube SteREO Discovery to equally split the view for teaching purposes, custom made Peltier cooling device, metal pins and dissection tools. (B–F) Steps for the making of an aluminum T-clip taken under the stereomicroscope. (G) Final size of a T-clip in the range of 0.9 mm wide open.

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  Figure 2. SEPN1 PCR genotyping representation Representative results of a SEPN1 PCR genotyping samples of a cohort of seven weaned mice run on agarose gel (1.5%). SEPN1 WT allele refers to the WT 682 bp amplicon, SEPN1 KO allele refers to the KO 209 bp amplicon. Ctrl SEPN1 WT refers to a known SEPN1 WT sample, Ctrl SEPN1 KO refers to a known SEPN1 KO sample, Ctrl SEPN1 HET refers to a known SEPN1 HET sample, Ctrl- refers to a blank (a PCR sample without DNA) and Marker 100 bp is a DNA ladder.

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  Figure 3. Peltier cooling device Custom made cooling system designed to keep the 60 mm Petri dish at low temperature during single fiber dissection. The system is composed of a Peltier device, temperature sensors and an Arduino to be the PID controller (A). A 3D printed aluminum circular circuit (B and C) connected to a water chiller removes the excessive heat from the lower Peltier side. The system allows background illumination as both the Peltier and the cooling aluminum components are circular. Designed using Autodesk Fusion 360 licensed to LN.

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  Figure 4. Single fiber T-clip placement scheme The scheme is showing the proper fiber placement when clipping on aluminum T-clips. Fiber should be centered, straight and with an overlap of about the same width of the clip body. Designed using PowerPoint and BioRender licensed to LN (University of Padova).

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  Figure 5. Single skinned fiber mounted on the Aurora 1410 permeabilized fiber setup (A) A single fiber mounted on the aluminum T-clips and on the setup, on the left the force tensiometer hook while on the right is the motor hook. The blue color at each end is due to the crosslinking procedure described. (B) Sarcomeres of a permeabilized fiber visualized on the microscope using the Aurora camera system.