Muscles were placed into 30% sucrose solution for cryoprotection before being embedded in optimal cutting temperature medium (OCT) and frozen in liquid nitrogen cooled isopentane. 10 μm sections were cut using a CM-3060S cryostat (Leica) and collected on charged glass slides. Histology was performed as previously described (Schmidt et al., 2017 (link), 2018 (link)) and included primary antibodies for rat anti-mouse CD31 (BioRad, Hercules, CA), rabbit anti-mouse dystrophin (BioRad, Hercules, CA), rabbit-anti mouse laminin (Sigma-Aldrich, St. Louis, MO), MyHC types I (BA-D5), IIa (SC-71), and IIb (BF-F3; Developmental Studies Hybridoma Bank, University of Iowa). Samples were mounted using Vectashield hard mount medium (Vector Labs) and imaged with an Evos FL auto microscope (Thermo Fisher Scientific, Waltham, MA).
Cm 3060s cryostat
Manufactured by Leica
The Leica CM-3060S is a cryostat, a device used for cutting thin sections of frozen biological samples. It provides precise temperature control to maintain the samples at the desired cryogenic temperature during the sectioning process.
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Lab products found in correlation
Rabbit anti mouse laminin, by Merck Group (1 mentions)
Evos fl auto microscope, by Thermo Fisher Scientific (1 mentions)
Sc 71, by Developmental Studies Hybridoma Bank (1 mentions)
Bf f3, by Developmental Studies Hybridoma Bank (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
2 protocols using cm 3060s cryostat
1
Cryosectioning and Immunohistochemistry of Skeletal Muscle
2
Aorta Immunohistochemistry in WT and PCMT1 Mice
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Aorta from 8-month-old WT and PCMT1 +/-mice were collected and fixed with 4% paraformaldehyde at 4°C for 24h. The aorta tissues were washed with PBS and supplemented with 15% sucrose then 30% sucrose over 1 day. Aortas were embedded in OCT compound with dry ice. A Leica CM3060S Cryostat was used to prepare 10μm cross sections of aorta that were mounted on Fisherbrand Superfrost plus Microscope slides. The slides were kept in warm PBS for 20min to remove OCT before being permeabilized and treated for 1h with blocking buffer (2.5% normal Goat serum, 1% BSA in 0.5%PBST) prior to addition of primary antibodies (isoDGR [1:200] and CD68 [1:200]) overnight at 4 °C. The slides were then incubated with secondary antibodies conjugated to AlexaFluor 488 and 594 for 1h at RT. DAPI staining for 30min was used to visualize cell nuclei.
After staining, the slides were washed with 1X PBS and mounted with aqueous mounting media.
Images were acquired using a Zeiss LSM710 confocal microscope.
After staining, the slides were washed with 1X PBS and mounted with aqueous mounting media.
Images were acquired using a Zeiss LSM710 confocal microscope.
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