Muscle strips were stretched to 150 % of their resting length (approximating Lo) and flash-frozen in melting isopentane. Serial transverse sections of DIAm strips were cut at 10 μm using a Reichert Jung Frigocut 2800 Cryostate (Reichert Microscope Services, Depew, NY, USA) and immunoreacted with the following antibodies specific to different MyHC isoforms: MyHCSlow (BA-F8, 1:3 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA), MyHC2A (SC-71, 1:3 dilution; Developmental Studies Hybridoma Bank) and Laminin (Sigma L9393, 1:200 dilution; Sigma-Aldrich, St. Louis, MO). In adjacent sections, MyHC2X (6H1, 1:1 dilution; Developmental Studies Hybridoma Bank) were incubated together, with the pattern of staining allowing the distinguishing of DIAm fibre types in a manner extensively used and previously validated (Schiaffino et al. 1989 (link), Prakash and Sieck 1998 (link), Elliott et al. 2016 (link)). Muscle cross-sections were imaged using a 20x oil-immersion objective (NA 1.0) on an Olympus FV2000 laser confocal microscope (Olympus America, Melville, NY) in a manner identical to past rat reports (Elliott et al. 2016 (link), Khurram et al. 2018a , Khurram et al. 2018b (link)).
Fv2000 laser confocal microscope
Manufactured by Olympus
Sourced in Japan
The FV2000 laser confocal microscope is a high-performance imaging system designed for a wide range of applications in life science research. The core function of the FV2000 is to provide high-resolution, optical sectioning of samples using laser excitation and sensitive detection technologies.
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Lab products found in correlation
Myhcslow ba f8, by Developmental Studies Hybridoma Bank (1 mentions)
Myhc2a sc 71, by Developmental Studies Hybridoma Bank (1 mentions)
Laminin sigma l9393, by Merck Group (1 mentions)
Dihydroethidium, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride, by Merck Group (1 mentions)
Compound c, by Selleck Chemicals (1 mentions)
Phospho ampk, by Abcam (1 mentions)
Ml385, by Selleck Chemicals (1 mentions)
2 protocols using fv2000 laser confocal microscope
1
Immunohistochemical Analysis of Diaphragm Muscle Fiber Types
2
Asiaticoside Attenuates Cardiac Dysfunction
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Asiaticoside (C48H78O20, ≥95%), triphenyl tetrazolium chloride, and 4′,6‐diamidino‐2‐phenylindole dihydrochloride were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Enzyme‐linked immunosorbent assay (ELISA) kits for detecting lactate dehydrogenase (LDH) and connective tissue growth factor (CTGF) levels were purchased from the Institute of Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Primary antibodies against AMPK, phospho‐AMPK, Nrf2, heme oxygenase 1 (HO‐1) and Beclin1, Atg5, LC3B, were purchased from Abcam (Cambridge, MA, USA). Primary antibodies against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) were purchased from Cell Signalling Technology (Boston, MA, USA). Goat antirabbit and goat antimouse secondary antibodies were purchased from Zhongshan Company (Beijing, China). Dihydroethidium, which detects the generation of reactive oxygen species (ROS) in cardiac tissues, was purchased from Invitrogen (Carlsbad, CA, USA). Compound C (CC) and ML‐385 were purchased from Selleck Chemicals (Houston, TX, USA).
The high‐resolution ultrasound imaging system (D700, Vinno, Suzhou, China) was purchased from Vinno Company, and the FV2000 laser confocal microscope was purchased from Olympus in Japan.
The high‐resolution ultrasound imaging system (D700, Vinno, Suzhou, China) was purchased from Vinno Company, and the FV2000 laser confocal microscope was purchased from Olympus in Japan.
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