ferentiation promote attachment of primary dissociated skeletal muscle cells, even though the ECM proteins vitronectin, 
laminin and tenascin-C were included in the media. Here we describe a method for decellularizing skeletal muscle that preserves most of the ECM molecules, including the proteoglycans. This decellularised skeletal muscle matrix was solubilized and examined for its ability to support C2C12 myoblast proliferation and differentiation under serum free culture conditions. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667322 The serum-free medium used was one that we developed specifically for C2C12 cell growth and differentiation on fibronectin substrates, and enabled us to assess the affect of the ECM on myoblast proliferation and differentiation in the absence of the confounding variables present in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666601 serum. The present study indicated that the complex muscle matrix supported myoblast differentiation, with differentiation occurring earlier on this matrix than on collagen I to result in the formation of well aligned myotubes. Under serum free conditions the myoblasts secreted and organised their own ECM when placed on a substrate and this endogenous matrix will also contribute to the myoblast’s local microenvironment. On 3D muscle matrices, in serum-free conditions, C2C12 cells aligned and orientated in patterns corresponding to the structure of the underlying muscle matrix. This suggests that although “generalised” ECM scaffolds can assist in muscle repair, the correct muscle ECM may [Lys8]-Vasopressin facilitate better cell organisation and differentiation. Materials and Methods Muscle sampling & ethics Female C57Bl/6J mice aged 3 months were obtained from the Animal Resource Centre and were acclimated for one week following delivery. All procedures were carried out in strict accordance with the guidelines of the National Health and Medical Research Council of Australia and were approved by the Curtin Animal Ethics Committee. Mice were anesthetized with 2% isoflurane and euthanized by cervical dislocation. Rats euthanized by intraperitoneal overdose of pentobarbitone sodium were obtained from Dr Christine Cooper, Curtin University. Following euthanasia, quadriceps muscles from both legs of the mice and rats were removed. Muscles were either snap frozen in liquid nitrogen, or embedded for cryosectioning and frozen using isopentane cooled with liquid nitrogen. All samples were stored at -80C. Decellularization of skeletal muscle sections 10 m thick frozen sections were cut using a cryostat, mounted on silanated glass slides and decellularized using either trypsin, SDS or phospholipase A2 . Unless otherwise stated, all chemicals and solutions used in these processes were purchased from Sigma Aldrich. DNA in muscle sections was visualized using Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole and images were captured using a Zeiss Axioskop fluorescent microscope and Spot Advanced software. Immunohistochemistry–muscle sections Frozen muscle sections were thawed and fixed in 4% paraformaldehyde/PBS for 10 min and blocked in 10% FBS/1% bovine serum albumin in PBS overnight at 4C. Sections were washed 3 times with PBS and incubated for 2 h in primary antibody diluted in blocking solution, before being washed with PBS and incubated in secondary antibody diluted in blocking solution. Sections were then washed and mounted in Vectashield. Images were captured with a Zeiss Axioskop fluorescent microscope using Spot Advanced software. Images were acquired at the same gain and exposure se
