Supplementary Materials Supplementary Material supp_137_20_3489__index. permits selective labeling of slow motor

Supplementary Materials Supplementary Material supp_137_20_3489__index. permits selective labeling of slow motor units and reveals their composition. Overexpression of the transcriptional co-regulator PGC1 in muscle fibers, which converts them to a slow phenotype, leads to an increased frequency of SV2A-positive motor nerve terminals, indicating a fiber type-specific retrograde influence of muscle fibers on their innervation. This retrograde influence must be integrated with known anterograde influences in order to understand how motor units become homogeneous. gene was obtained from Incyte Genomics (St Louis, MO, USA). This clone spanned 165 kb with the gene located near the center. A recombineering method (Lee et al., 2001) was used to replace the segment between 78 bp upstream and 496 bp downstream of the initiator codon with a cDNA encoding tamoxifen-inducible Cre recombinase T2 (CreER) (Feil et al., 1997). Transgenic mice harboring the recombined BAC clone were generated by oocyte injection and maintained on a C57/B6 background. SV2ACreER mice were crossed to Thy1-STOP-YFP mice, which we generated previously (Buffelli et al., 2003). To activate CreER, 0.5 mg tamoxifen (50 l of a 10 mg/ml solution in 10:1 corn oil:ethanol) was injected intraperitoneally. MCK-PGC1 transgenic mice were generated as described (Lin et al., 2002). Immune-deficient (SCID) mice were obtained from Jackson Laboratories (#001303). Experiments on wild-type mice used C57/B6 and CD-1 animals interchangeably; no differences between these strains were noted. Histology Antibodies Primary antibodies were anti-SV2A [AB15224 from Millipore and EGI916 from T. Sudhof, Stanford University (Janz and Sudhof, 1999)], anti-SV2B (EGI916 from T. Sudhof), anti-SV2C (U1129 from T. Sudhof), anti-MyHC I [A4840 from Developmental Studies Hybridoma Bank (DSHB) and NCLslow from Leica Microsystems/Novacastra Laboratories], anti-MyHC IIA (2F7 and SC-71 from DSHB), anti-pan MyHC except IIX (BF35 from DSHB), anti-MyHC IIB (BFF3 from DSHB), anti-actinin alpha 3 (Abcam) and anti-GFP (Millipore). Alexa-conjugated Panobinostat kinase activity assay secondary antibodies and -bungarotoxin (BTX) were from Invitrogen. Cross-sections Muscles were dissected, rinsed in phosphate-buffered saline (PBS) pH 7.4, embedded in Tissue Freezing Medium (Electron Microscopy Sciences), frozen in melting 2-methyl butane in liquid nitrogen, and cross-sectioned at 8 m on a cryostat. Sections were allowed to thaw for 5 minutes and subsequently blocked with 1% normal goat serum, 4% bovine serum albumin and 0.1% Triton X-100 (GAT) for 1 hour. Sections were then stained with primary antibodies overnight at 4C followed by incubation with secondary antibodies and Alexa-conjugated BTX for 1 hour at room temperature, then mounted in Fluoro Gel (Electron Microscopy Sciences). Images were taken with an Apotome microscope (40 objective, Zeiss) or a Fluoview1000 confocal microscope (1.45 NA objective lens, Olympus). Levels were adjusted in Photoshop (Adobe) and individual channels were combined to generate color images. Longitudinal sections Muscles were dissected, rinsed in PBS, fixed in 4% Rabbit Polyclonal to CPN2 paraformaldehyde (PFA) in PBS for 1 hour, incubated in 30% sucrose at 4C overnight, and frozen as above. Longitudinal areas had been cut at 12 m, refixed in methanol at C20C for ten minutes, and clogged for one hour in GAT. Sections were stained Panobinostat kinase activity assay then, analyzed and imaged as referred to over. Whole-mounts and vibratome areas Mice had been perfused with 4% PFA, muscles were isolated then, post-fixed in ice-cold 2% PFA for thirty minutes at 4C, and blocked in GAT plus 0 overnight.1 M glycine and 0.02% sodium azide. Muscle groups were in that case incubated for one day each with major antibodies and extra Alexa-conjugated in addition antibody BTX. Panobinostat kinase activity assay After cleaning in PBS, muscle groups had been mounted in.