Computational protocol: Differential myofiber-type transduction preference of adeno-associated virus serotypes 6 and 9

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Protocol publication

[…] After AAV injection, mice were imaged on a weekly basis for a period of 4 weeks using the Maestro™ in vivo fluorescence imaging system (Xenogen product from Caliper Life Sciences, Hopkinton, Massachusetts, USA) according to the manufacturer’s instructions. Mice were anesthetized with a continuous flow of 2 % isoflurane prior to imaging, and GFP fluorescence was acquired with an acquisition time ranging from 30 to 90 s depending upon the fluorescent signals from the AAV-injected TA muscles.Four-week post-injection mice were sacrificed by cervical dislocation, and TA muscles were collected and immersed in liquid nitrogen cold isopentane for about 30–45 s and then stored at −80 °C for further processing. Transverse cryosections of 10-μm thick were made with a cryostat (Leica CM3050S) and pasted on superfrost plus glass slides (Menzel-Gläser; Thermo scientific) from four alternating parts across the muscle (Additional file : Figure S1). Additional tissue from the intervening areas was used for RNA isolation, ensuring representation of the whole muscle during data collection. Histological analyses of the muscle sections included hematoxylin and eosin staining [] and immunofluorescence for myofiber typing. The untreated cryosections were directly incubated with a primary antibody to Laminin (1:1000; ab11575, Abcam). When indicated, a supernatant of hybridoma 6H1 (1:5, Developmental Studies Hybridoma Bank (DSHB), USA), detecting MyHC-2x was co-incubated. These antibodies were detected with secondary anti-rabbit-alexa-647 and anti-mouse-alexa-546 (Molecular Probes, Invitrogen). A mixture of monoclonal antibodies to MyHC-1, MyHC-2a, and MyHC-2b isotypes (hybridoma BA-D5, SC-71 and BF-F3, respectively (DSHB)), conjugated with alexa-350, alexa-594, and alexa-488, respectively was generated as describe before []. Antibody production from hybridomas and fluorophore conjugation was carried out as described earlier []. All antibody incubations were carried out in PBS containing 0.05 % tween (PBST) and 5 % dry milk, and washes were carried out with PBST. Slides were mounted with Aqua Polymount (Polyscience). Incubation with only anti-mouse conjugated secondary was carried out to exclude nonspecific binding to GFP positive myofibers. Imaging of GFP was carried out in untreated sections that were directly mounted with Polymount containing 4′,6-diamidino-2-phenylindole (DAPI). Imaging was carried out with either DM5500 or TCS-SP5 (confocal) microscopes (Leica) using LAS AF software versions: 2.3.6 (DM5500) and 2.5.1.6757 (TCS-SP5), respectively.Image quantification was carried out with ImageJ, version 1.48 []. Per myofiber mean fluorescence intensity (MFI) and cross-sectional area (CSA) were documented after segmenting sections based on Laminin staining. Subsequently, MFI values were corrected for background and normalized for a fluorophore constant, which was determined from unbound fluorochrome per microscope and camera combination: (DM 5500: 430 nm = 0.735; 488 nm = 0.067; 596 nm = 0.172 and SP5: 488 nm = 0.003; 546 nm = 3.90). [...] Fold changes and statistical significance of mRNA were determined with the Student’s t test in GraphPad Prism version 6.02. P values <0.05 were considered statistically significant. Statistical analyses of myofibers were carried out on natural log transformed values of the MFI (normalized to the fluorophore constant). A correlation between MFI of GFP and MyHC isotypes was assessed in a multivariate linear regression model with GFP MFI as a dependent variable and MyHC MFIs as independent variables. In a second multivariate model, CSA was added into the first model as an independent variable. Both models were stratified based on AAV serotypes. In addition, Pearson correlation was used to assess correlations between CSA, GFP, and the MFI of each MyHC isotype. Analyses were performed with IBM SPSS Statistics version 20.0. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Muscular Dystrophies