A method for preparing skeletal muscle fiber basal laminae

Previous attempts to prepare skeletal muscle basal laminae (BL) for ultrastructural analyses have been hampered by difficulties in successfully removing skeletal muscle proteins and cellular debris from BL tubes. In the present study we describe a two phase method which results in an acellular muscle preparation, the BL of which are examined by light, transmission electron, and scanning electron microscopy. In the first phase, excised rat extensor digitorum longus muscles are subjected to x‐radiation and then soaked in Marcaine to inhibit muscle regeneration and to destroy peripheral muscle fibers. The muscles are then grafted back into their original sites and allowed to remain in place 7–14 days to allow for maximal removal of degenerating muscle tissue with minimal scar tissue formation. In the second phase, the muscle grafts are subjected sequentially to EDTA, triton X‐100, DNAase, and sodium deoxycholate to remove phagocytizing cells and associated degenerating muscle tissue. These procedures result in translucent, acellular muscle grafts which show numerous empty tubes of BL backed by endomysial collagenous fibers. These preparations should be useful for morphological analyses of isolated muscle BL and for possible in vitro studies by which the biological activity of muscle BL can be examined.

[1]  M. Farquhar.  The Glomerular Basement Membrane , 1981 .

[2]  E. Turley,et al.  The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices. , 1985, Developmental biology.

[3]  E. Carlson,et al.  Ultrastructure of isolated basement membranes in the acellular human renal cortex. , 1983, Renal physiology.

[4]  B. Carlson.  A quantitative study of muscle fiber survival and regeneration in normal, predenervated, and Marcaine-treated free muscle grafts in the rat , 1976, Experimental Neurology.

[5]  E. Carlson,et al.  Native banded collagen fibrils in the glomerular mesangial matrix of normal human and laboratory animals. , 1981, Journal of ultrastructure research.

[6]  R. Vracko,et al.  BASAL LAMINA: THE SCAFFOLD FOR ORDERLY CELL REPLACEMENT , 1972, The Journal of cell biology.

[7]  F. Hansen-Smith.  Formation of acetylcholine receptor clusters in mammalian sternohyoid muscle regenerating in the absence of nerves. , 1986, Developmental biology.

[8]  A. K. Gulati.  Basement membrane component changes in skeletal muscle transplants undergoing regeneration or rejection , 1985, Journal of cellular biochemistry.

[9]  U J McMahan,et al.  Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites , 1978, The Journal of cell biology.

[10]  M. Karnovsky,et al.  POROUS SUBSTRUCTURE OF THE GLOMERULAR SLIT DIAPHRAGM IN THE RAT AND MOUSE , 1974, The Journal of cell biology.

[11]  E. Carlson,et al.  SEM and TEM analyses of isolated human retinal microvessel basement membranes in diabetic retinopathy , 1990, The Anatomical record.

[12]  H. Latta.  The glomerular capillary wall , 1970 .

[13]  D. Bader.  Density and distribution of α-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle , 1981, The Journal of cell biology.

[14]  J. Sanes,et al.  Cholinesterase is associated with the basal lamina at the neuromuscular junction , 1978, Nature.

[15]  R. Bischoff.  Proliferation of muscle satellite cells on intact myofibers in culture. , 1986, Developmental biology.

[16]  E. Carlson.  Topographical specificity in isolated retinal capillary basement membranes: a high-resolution scanning electron microscope analysis. , 1988, Microvascular research.

[17]  R. Vracko,et al.  Basal lamina scaffold-anatomy and significance for maintenance of orderly tissue structure. , 1974, The American journal of pathology.

[18]  E C Carlson,et al.  Ultrastructural analysis of major basement membrane types in rhesus monkey Macaca mulatta acellular renal cortex. , 1986, Acta anatomica.

[19]  M. Farquhar.  The Glomerular Basement Membrane A Selective Macromolecular Filter , 1991 .

[20]  R. Coggeshall,et al.  A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY , 1965, The Journal of cell biology.

[21]  J. Brody,et al.  Structural features of alveolar wall basement membrane in the adult rat lung , 1981, The Journal of cell biology.

[22]  E. Carlson,et al.  SEM studies of acellular glomerular basement membrane in human diabetic glomerulopathy , 1986, The Anatomical record.

[23]  B. Carlson.  The regeneration of minced muscles. , 1972, Monographs in developmental biology.

[24]  E. Carlson,et al.  A topographical (SEM) analysis of acellular glomerular mesangial matrix in situ. , 1983, Journal of ultrastructure research.

[25]  A. Zalewski,et al.  An immunofluorescent analysis of lectin binding to normal and regenerating skeletal muscle of rat , 1985, The Anatomical record.

[26]  P. Sargent,et al.  Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve , 1979, The Journal of cell biology.

[27]  E. Carlson.  Fenestrated subendothelial basement membranes in human retinal capillaries. , 1989, Investigative ophthalmology & visual science.