F view 2 ccd camera

MVN tissue sections of mice were baked at 60 °C for 20 min, soaked in xylene for 15 min, and dehydrated with graded ethanol (100, 95, 90, 85, and 80%). After that, each section was dripped with 3% H₂O₂ and soaked at room temperature for 10 min to block endogenous peroxidase, followed by antigen repair for 10 min. The samples were incubated with normal goat serum blocking solution (Sangon, Shanghai, China) at room temperature for 20 min and then with primary antibodies against NeuN (ab207279, 1:500, Abcam) and Annexin (ab108194, 1:50, Abcam) overnight at 4 °C. Cells were incubated with Alexa-Fluor488 or Alexa-Fluor594 coupled secondary antibodies (Invitrogen), and the nuclei were stained with 0.1 µg/ml DAPI. The cells were analyzed using an Olympus BX61 microscope equipped with an F-View II CCD camera. In each experiment, images were acquired using constant camera settings and analyzed via the Cell P (Ommpus Soft Imaging Solutions) program. At least 15 visual fields were randomly selected for each sample.

Human primary myoblasts were cultured in DM or GM for up to 96 h as described above and cells were fixed using 4% PFA. The localization of F-actin was determined using Phalloidin Alexa488 (Molecular Probes, Life Tech., Darmstadt, Germany), the cell membrane was stained with wheat germ agglutinin conjugated with Alexa594 and the nuclei with Hoechst 33342 according to [11 (link)]. Representative photos were made using the confocal microscope Nikon Eclipse E1000-M or using an Olympus BX-51 fluorescent microscope (Olympus, Hamburg, Germany) equipped with an F-View II CCD camera (Soft Imaging System, Stuttgart).

FISH for quantitation of MYC and chromosome 8 centromere (CEP8) was performed on 5 μm FFPE tissue sections using the ZytoLight SPEC MYC/CEN 8 Dual Color Probe (Zytomed Systems GmbH, Berlin, Germany). Deparaffinization, denaturation and hybridization were done according to the manufacturer's instructions. For denaturation and hybridization a StatSpin Hybridizer instrument (cat # S2450, Dako, Hamburg, Germany) was used. Hybridized slides were counterstained with Vectashield Antifade Mounting Medium with DAPI (H-1200, Vector Laboratories, Inc., Burlingame, CA, USA) and then analyzed using an Olympus BX41 fluorescence microscope (Olympus Deutschland GmbH, Hamburg, Germany) with optical filters for DAPI, SpectrumGreen and SpectrumOrange (Olympus) with a UPlanSApo 60x objective (oil, numerical aperture 1.35; Olympus). The microscope was connected to an F-View II CCD-Camera (Soft Imaging System GmbH, Muenster, Germany). Cell^F software (Olympus Soft Imaging Solutions GmbH, Muenster, Germany) was used to acquire representative images with each filter. Sixty interphase nuclei were counted per sample. Following previous publications [23 (link)–25 (link)] the following signal patterns were defined:

Fluorescent images were taken on epi-fluorescence microscopes (Olympus BX61 and BX63). Twelve-bit images were acquired using 20× objective lenses with an F-View II CCD camera (Olympus Soft Imaging Solutions, Muenster, Germany) or an XM10 Monochrome camera (Olympus) at 1376 × 1032 (Olympus BX61) or 1376 × 1038 (Olympus BX63) pixel resolution. Images were taken at the best-focus position, where the staining signals were brightest in the section.
Synapse formation in the CA3 region was assessed using immunostaining for VGLUT1 and VGAT. Images were taken from the CA3a and CA3b regions of CA3 (see Figure 1B). The size and density of stained puncta were quantified and analyzed using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). The staining intensity in the fimbria, a myelinated tract of axons located in the medial region of CA3, was calculated and used for background subtraction from each image. Neurogenesis within the DGC layer was assessed using immunostaining for DCX, a marker for immature neurons, and Prox1, a marker for DGCs. The number of DCX-positive cells was divided by the number of Prox1-positive DGCs or that of DAPI-positive DGCs. Quantification was done from both the upper and lower blades of the DGC layer.