Longissimus thoracis muscles obtained from 40-day-old piglets were frozen in dry-ice acetone (−78°C), and cryosections were generated using a cryostat microtome (Sakura Finetek). To analyze the distribution of skeletal muscle fiber types, the sections were immunostained with anti-slow (clone M8421, 1:400; Sigma) and anti-fast (clone M4276, 1:200; Sigma) myosin heavy-chain monoclonal antibodies, which are specific markers of type I and type II myofibers, respectively. Histofine Simple Stain MAX PO (M) (Nichirei) was used as the secondary antibody. The proportion of each fiber type in each section of the longissimus thoracis muscle was determined using a digital microscope (VHX-5000; Keyence).
Cryostat microtome
Manufactured by Sakura Finetek
The Cryostat Microtome is a laboratory instrument used to cut thin, frozen tissue samples for microscopic examination. It maintains a controlled low-temperature environment to preserve the integrity of the samples during the sectioning process.
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2 protocols using cryostat microtome
1
Skeletal Muscle Fiber Typing in Piglets
2
Neural Stem Cell Transplantation in Mice
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All mouse experiments were performed in Kunming Medical University and the protocol was approved by Kunming Medical University Committee for animal welfare, and the methods were carried out in accordance with the approved guidelines. NESCs were infected by retrovirus containing GFP and performed clonal expansion. Single-derived GFP positive colonies were selected to continually expand. Five GFP positive cell lines were established and one cell line was randomly used for following transplantation.
About 2 μl (2 × 105/μl) of trypsin-dissociated cells in PBS were injected into the ventricles of eleven new born SCID mouse (postnatal 2-5 days). 0.1% food dye was applied to confirm the injection sites. One to three months after transplantation, animals were anesthetized and perfused with 4% paraformaldehyde. Brains were then extracted, post-fixed in 4% PFA (2–3 days) and dehydrated in sucrose solution (10%, 20% and 30%, respectively). The transplanted sites were eventually sectioned on a cryostat microtome at 18 μm after embedding in O.C.T (Sakura-Finetek) and stained with GFP and the neuron or astrocyte antibodies. All antibodies, sources and dilutions are listed in table (Fig. S3B ).
About 2 μl (2 × 105/μl) of trypsin-dissociated cells in PBS were injected into the ventricles of eleven new born SCID mouse (postnatal 2-5 days). 0.1% food dye was applied to confirm the injection sites. One to three months after transplantation, animals were anesthetized and perfused with 4% paraformaldehyde. Brains were then extracted, post-fixed in 4% PFA (2–3 days) and dehydrated in sucrose solution (10%, 20% and 30%, respectively). The transplanted sites were eventually sectioned on a cryostat microtome at 18 μm after embedding in O.C.T (Sakura-Finetek) and stained with GFP and the neuron or astrocyte antibodies. All antibodies, sources and dilutions are listed in table (
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