Identification of skeletal muscle mutations in tail snips from neonatal mice using immunohistochemistry.

Naturally occurring or laboratorygenerated genetic modifications of striated muscle in mice have been linked to cardiomyopathies or muscular dystrophies (1–4). Such mutations are generally recognized by recombinant DNA techniques, especially PCR, but uncertainties about the genetic locus of particular mutations and variations in DNA sequences among different strains of mice may limit the usefulness of this approach. We investigated the possibility of using tail snips, typically obtained for PCR analysis, for immunofluorescence assays of muscle proteins. We report here that skeletal muscle fibers present in the distal 1-cm-end of the tail of the neonatal mouse can be assayed using immunohistochemistry to identify desmin null (5), dysferlin null (6), and dystrophin null muscles. Identification of these muscles by immunofluorescent assays allows phenotypic conformation of genetic screening of mutant mice, facilitating evaluation of muscle from only those animals expressing the genotype of interest. Standard tail snips, approximately 1 cm in length, were collected from young mice (<4 weeks of age) and used for PCR or immunofluorescence analysis or both. The tails were swabbed with alcohol, and sterile surgical scissors were used to obtain tissue. For immunolabeling, tissue (≤5 mm) was mounted with optimal cutting temperature (OCT) medium and snap-frozen in pentane-cooled liquid nitrogen. Serial cross-sections, 10 μm in thickness, were prepared with a cryostat, collected on glass slides (Superfrost® Plus; VWR, West Chester, PA, USA), incubated with phosphatebuffered saline (PBS) containing 3% bovine serum albumin (BSA) for 1 h, and labeled with primary antibodies diluted in the same solution to muscle myosin (mouse 1:500; Sigma-Aldrich, St. Louis, MO, USA) and desmin (rabbit 1:100; Neomarkers, Fremont, CA, USA), a muscle-specific intermediate filament protein. Other serial sections were labeled with antibodies to desmin and dystrophin (mouse 1:5; Novocastra, Newcastle upon Tyne, UK) or desmin and dysferlin (mouse 1:100; Novocastra). Sections were washed three times for 10 min with PBS, then incubated in speciesspecific secondary antibodies coupled to Alexa dyes (Invitrogen, Carlsbad, CA, USA). Samples were mounted in VECTASHIELD® (Vector Laboratories, Burlingame, CA, USA) and covered with glass coverslips (VWR), before examination under epifluorescent optics (Zeiss Axioskop® 50, Carl Zeiss, Poughkeepsie, NY, USA). All labeling of wild-type tail muscle was specific, as judged by the use of appropriate preimmune serum. Identification of skeletal muscle mutations in tail snips from neonatal mice using immunohistochemistry