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)).
Laminin sigma l9393
Manufactured by Merck Group
Laminin (Sigma L9393) is a purified extracellular matrix glycoprotein derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is a high-molecular-weight protein composed of three different polypeptide chains: alpha, beta, and gamma. Laminin is a key structural component of the basement membrane and plays a critical role in cell adhesion, differentiation, migration, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Myhcslow ba f8, by Developmental Studies Hybridoma Bank (2 mentions)
Myhc2a sc 71, by Developmental Studies Hybridoma Bank (2 mentions)
Fv2000 laser confocal microscope, by Olympus (1 mentions)
Cd31 pecam 1, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
2 protocols using laminin sigma l9393
1
Immunohistochemical Analysis of Diaphragm Muscle Fiber Types
2
Skeletal Muscle Fiber Typing and Vascular Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
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All frozen skeletal muscles were placed in a Leica cryostat and transverse sections of 10 μm were performed. As previously described by Gill et al.15 (link), sections were blocked for 45 min in 10% goat serum diluted in PBS 1X solution and incubated overnight at 4 °C in primary antibodies for the following Myosin heavy chain (MyHC) isoforms: MyHCSlow (BA-F8, 1:100 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA), MyHC2A (SC-71, 1:100 dilution; Developmental Studies Hybridoma Bank), and Laminin (Sigma L9393, 1:200 dilution; Sigma-Aldrich, St. Louis, MO). Secondary antibodies were then incubated at a 1:2000 dilution, using Alexa Fluor Goat anti-Mouse IgG2b 647 (for MyHCSlow), Goat anti-Mouse IgG1 555 (for MyHC2A) and Goat anti-Rabbit 594 (for Laminin)15 (link). CD 31 (PECAM-1, 1:100; Thermo Fisher Scientific, Inc., MA) antibody was incubated with Laminin antibody and Alexa Fluor Goat anti-Rat 488 and Goat anti-Rabbit 594 were applied at 1:2000 dilution.
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