Axio cell observer z1

Skeletal muscle cells were fixed with paraformaldehyde for 90 min after treatment with acteoside or vehicle solution. Myotubes were immunostained with the rabbit polyclonal anti-desmin antibody (1:250; Covance Inc.), followed by the Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (1:300). Images, 10–13 per well, were captured using a fluorescent microscope (Axio Cell Observer Z1, Carl Zeiss), at a field of 432 μm × 323 μm or 863 μm × 645 μm. Desmin-positive cells were counted using MetaMorph software.

Immunofluorescence and double ISH images of cryosections at thoracic level of the developing spinal cord were acquired on a Zeiss Axio Cell Observer Z1 confocal microscope with the Zeiss AxioVision Rel. 4.8 software and processed with Adobe Photoshop CS5 software. Double ISH images were also acquired using an EVOS^® FL Auto Imaging System (ThermoFisher Scientific) and related software. Quantifications were performed on red or green or blue layer of acquired confocal images and double or triple labeling cells were processed by subtractive method (Francius and Clotman, 2010 (link)). For each embryo (n ≥ 3), both sides of three sections at thoracic level were quantified using the count analysis tool of Adobe Photoshop CS5 software.
Raw data were exported from Adobe Photoshop CS5 software to SigmaPlot v12.3 software and processed in order to generate histogram figures. All data were analyzed and histograms were made with SigmaPlot software. Adequate statistical tests were applied based on the number of comparisons and on the variance in each group. For analysis of cell quantification based on comparison of two groups (control or mutant), standard Student’s t-tests were performed. Quantitative analyses were considered significant at p < 0.05. Three asterisks (***) indicate p < 0.001.

After treatment with the CM, recombinant pyruvate kinase isoform M2 (PKM2; 0.01, 0.1, 1, and 10 ng/mL), or vehicle solution, neurons were fixed with paraformaldehyde for 90 min. Axons were immunostained with the mouse monoclonal anti-phosphorylated neurofilament-H (pNF-H) antibody (1:250; Covance Inc., Princeton, NJ). Neuronal cell bodies were immunostained with the rabbit polyclonal anti–microtubule-associated protein 2 (MAP2) antibody (1:2000; Abcam, Cambridge, UK). As secondary antibodies, we used Alexa Fluor-488-conjugated goat anti-mouse immunoglobulin G (IgG) and Alexa Fluor-568-conjugated goat anti-rabbit IgG (1:300; Thermo Fisher Scientific). Nuclear staining with 1 μg/mL 4′6-diamino-2-phenylindole (DAPI) was performed at room temperature. Eight to 23 images per well were captured using a fluorescent microscope (Axio Cell Observer Z1; Carl Zeiss, Oberkochen, Germany), at a field of 432 μm × 323 μm or 863 μm × 645 μm. The length of pNF-H–positive axons was measured using MetaMorph software (Molecular devices, San Jose, CA), which automatically traces and measures neurite lengths. The total axonal length in a given image was divided by the number of MAP2- and DAPI-double positive cells to calculate the axonal length per neuron.