Ncl drp2

Immunohistochemical analysis involved the use of immunoperoxidase techniques on frozen sections. MANDYS110 clone 3H10, provided by the Wolfson Centre for Inherited Neuromuscular Disease, NCL-DYSB and NCL-DYS2 (Novocastra Laboratories, Newcastle on Tyne, UK) mouse monoclonal antibodies were used for dystrophin protein detection (1∶50 for each ones). Other primary antibodies used were IVD3₁A9 (1∶50, Developmental Studies Hybridoma Bank, Iowa City, IA), NCL-g-Sarc (1∶10, Novocastra Laboratories) and NCL-DRP2 (1∶60, Novocastra Laboratories) for alpha-sarcoglycan, gamma-sarcoglycan, and utrophin detection respectively, NCL-MHCd (1∶20, Novocastra Laboratories) for developmental myosin heavy chain isoform, anti-complement 5b-9 (1∶250, Calbiochem, Strasbourg, France) and anti-CD3 (1∶100, Dako, Glostrup, Denmark) for lymphocytes. Briefly, transverse cryosections were incubated in PBS with 5% normal goat serum (Dako) for 1 hour at room temperature. They were then incubated with primary antibody in 5% rat serum overnight at 4°C and with biotinylated secondary antibodies (E433, 1∶300, Dako) in PBS with 5% rat serum for 1 hour. Bound antibodies were detected either with streptavidin (P397; Dako) and DAB Liquid Substrate (Dako) for immunoperoxidase. Fiber type was determined using histochemical myosin-ATPase reaction after preincubation at pH 4.2, 4.35, and 10.4 as previously described [22] .

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)].