Characterization of Muscle Biopsies in French Bulldog

Histopathological and immunofluorescent characterizations of muscle biopsies from both Brittany spaniels were previously described [30 (link)]. Unfixed, chilled and formalin-fixed diagnostic muscle biopsy specimens were collected post-mortem from the biceps femoris, diaphragm and tongue muscles of the French bulldog and shipped by an overnight express service under refrigeration to the Comparative Neuromuscular Laboratory, University of California San Diego. Upon receipt, the unfixed muscle specimens were snap frozen in isopentane, pre-cooled in liquid nitrogen, and stored at −80 °C until further processed. Cryosections were evaluated using a standard panel of histochemical stains and reactions [31 ]. Additional cryosections were cut and stained for indirect immunofluorescence as previously described [32 (link)], using monoclonal antibodies against the rod (1:100, NCL-DYS1) and carboxy-terminus (1:100, NCL-DYS2) of dystrophin, utrophin (1:20, NCL-DRP2), and developmental myosin heavy chain for regenerating fibers (1:20, NCL-dMHC), all from Novocastra Laboratories, Newcastle, UK; a monoclonal antibody against caveolin 3 (1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and polyclonal antibodies against laminin α2 (1:200), α-sarcoglycan (1:200), and collagen VI (direct apply, monoclonal antibody 3G7), all gifts of Professor Eva Engvall [33 (link),34 (link)].