In 25 OSA patients and 20 controls muscle biopsies were obtained from the vastus lateralis muscle at the exact site of 1H-MRS by conchotome technique through an 8-mm skin and fascia incision after local anesthesia and disinfection, immediately shock-frozen in liquid N2-cooled isopentane, and stored at –80 °C. Transverse 7 µm-cryosections (cryostat microtome Hyrax C60, Carl Zeiss AG) were used for histochemical identification of fiber type 1, 2a, or 2x using the acid-sensitive myofibrillar adenosine triphosphatase (ATPase) staining (adenosine triphosphatase, Sigma-Aldrich Co LLC) after preincubation at pH 4.6 (5 minutes, room temperature) as previously described [54 (link)]. Fiber-type–specific cross-sectional area and composition (percentage of type 1, 2a, and 2x fiber count and overall area) were analyzed in rectangular tissue areas with more than 100 fibers by applying ImageJ (Scion Image, National Institutes of Health) to digitized images with 200-fold magnification obtained by the Zeiss Axio Imager.M2 microscope (Carl Zeiss AG combined with Axio-Cam HRc/AxioVision,).
Hyrax c60
Manufactured by Zeiss
Sourced in Germany
The Zeiss Hyrax C60 is a laboratory instrument designed for high-resolution X-ray diffraction analysis. It features a compact and robust design, providing stable and precise measurements of various samples. The core function of the Hyrax C60 is to perform X-ray diffraction analysis for the characterization of crystalline materials.
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3 protocols using hyrax c60
1
Muscle Fiber Analysis in OSA Patients
2
Fixation and Cryosectioning of Rat Eyes
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Anaesthetized rats were euthanized by intracardial injection of lethabarb (Virbac Pty.Ltd., NSW, Australia) immediately prior to tissue collection. For orientation, the dorsal region of the eye was marked with a tattoo pen, eyes were removed, the anterior components dissected, and the posterior eye cup placed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 30 min. The eye cups were subsequently cryoprotected in graded sucrose solutions (10, 20, 30% v/w) and snap frozen and stored at −80°C until use.
Frozen sections (14 μm) were cut on a cryostat (Hyrax C60, Zeiss, Germany), mounted onto poly-L-lysine coated glass slides (Polysine® Adhesion slides, Thermo Scientific, VIC, Australia) and stored at −80°C until use. All sections were cut from the dorsal to ventral region of the eye, as close to the optic disc as possible, to allow for consistency between eyes.
Frozen sections (14 μm) were cut on a cryostat (Hyrax C60, Zeiss, Germany), mounted onto poly-L-lysine coated glass slides (Polysine® Adhesion slides, Thermo Scientific, VIC, Australia) and stored at −80°C until use. All sections were cut from the dorsal to ventral region of the eye, as close to the optic disc as possible, to allow for consistency between eyes.
3
Muscle Biopsy Sampling and Analysis
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Muscle biopsies were obtained from 13 out of 15 MA and 10 out of 11 YG subjects from the VLat muscle using a conchotome of 6 mm width through an 8 mm skin and fascia incision after careful local anesthesia and desinfection. Exact biopsy localisation within the VLat was controlled by MRT 2 h after sampling (Fig 2B ). Muscle samples were immediately shock-frozen in liquid nitrogen-cooled isopentane and stored at -80°C. Serial transverse cryo-sections (6–7 μm) were cut in a cryostat microtome (Hyrax C60, Carl Zeiss AG, Oberkochen) at -20°C for histomorphometry of capillarisation and fiber composition.
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