Axioplan brightfield microscope

Human open muscle biopsies from two patients carrying the CNM- dynamin-2 mutation p.R465W, one patient carrying the CNM-dynamin-2 mutation p.R369Q, one patient with dystrophy harboring a mutation in dysferlin (dysferlinopathy), one patient with dystrophy harboring a mutation in dystrophin (dystrophinopathy) and one healthy control muscle were performed at the Centre de Référence de Pathologie Neuromusculaire Paris-Est, Institut de Myologie, GHU Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, GH Pitié-Salpêtrière, Paris, France, following written informed consent specially dedicated for diagnosis and research. All experimental protocols were approved by the ethics committee of the Centre de Référence de Pathologie Neuromusculaire Paris-Est, Institut de Myologie, GHU Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, GH Pitié-Salpêtrière, Paris, France. For conventional histochemical and immunocytochemical techniques see reference^{74 (link)}. Briefly, frozen muscle samples were stained with a specific anti-GLUT4 antibody and revealed by immunoperoxidase techniques (Roche-Benchmark). Digital photographs were obteined with a Zeiss AxioCam HRc linked to a Zeiss Axioplan Bright Field Microscope (Zeiss, Germany).

Conventional histochemical techniques were performed for hematoxylin and eosin (HE), Engel–Gömöri trichrome, cytochrome C oxidase (COX), nicotinamide adenine dinucleotide (NADH), menadione-linked αglycerophosphate dehydrogenase, and the immunostaining of fast myosin heavy chain (MHC-fast), ubiquitin, desmin, and dystophin. Digital photographs of each biopsy were obtained using a Zeiss AxioCam HRc linked to a Zeiss Axioplan Bright Field Microscope and processed using Axio Vision 4.4 software (Zeiss, Jena, Germany).

Posterior midguts of adult females were collected and fixed in 2.5% of glutaraldehylde in 0.1 M sodium cacodylate buffer pH 7.2 for at least 24 h at 4°C, followed by a post-fixation in 1% osmium tetroxide, 0.8% potassium ferricyanide and 2.5 mM calcium chloride in the same buffer for 1 h at room temperature. The dehydration steps were performed by incubations of 15 min for each acetone concentration (30%, 50%, 70%, 90% and 100%). Then the samples were gradually embedded in epoxy polybed resin (Polybed 812, Polysciences, Germany). Infiltration was performed by incubating samples with 1:3, 1:2, 2:3 of epoxy polybed resin:acetone for 24 h to each step. After that, samples were infiltrated with epoxy polybed resin 100%, incubated at room temperature for 4 h and then incubated at 60°C for 4 days to complete polymerization. Then, semi-thin sections (0.5 μm) were obtained, stained with toluidine blue and observed in a Zeiss Axioplan bright field microscope for histological analysis. Alternatively, for ultrastructural analysis, ultrathin sections were obtained with an ultramicrometer Ultracuts (Leica) collected in copper grids, stained in uranyl acetate for 20 min and lead citrate for 2 min, and sections were observed in a JEOL JEM1011 transmission electron microscope at Oswaldo Cruz Institute electron microscopy platform.