Eclipse te200 microscope

Cell proliferation index was evaluated by proliferating cell nuclear antigen (PCNA) immuno-staining. Cells were fixed in methanol at -20 °C for 15 min, while paraffin-embedded tissues were treated with 10mM Sodium citrate solution (pH 6) containing 0,05% Tween-20 (Sigma-Aldrich) to unmask antigens. Samples were incubated in blocking solution containing 10% Goat Serum (Sigma-Aldrich) in PBS for 30 min. Primary antibody (1:200, Sigma-Aldrich) was incubated for 1 h, followed by a suitable secondary antibody exposure (1:250, Alexa Fluor™) for 1 h. Nuclei were counterstained with 4′,6-diamidino-2- phenylindole (DAPI, Sigma-Aldrich). Samples were observed under the Eclipse TE200 microscope (Nikon). All steps were performed at room temperature, unless otherwise indicated. The number of immuno-positive cells was counted in 10 randomly selected fields at 100× total magnification. A minimum of 500 cells were scored in three independent replicates. The number of PCNA positive cells was expressed as a percentage of the total cell counted.

ESCs constitutively expressing histone H2B-GFP were provided by Dr Kat Hadjantonakis [37 (link)]. Mitomycin-inactivated mouse embryonic fibroblasts, which served as the feeder layer for ESCs, were plated on dishes coated with 1% gelatine (Sigma-Aldrich) in DMEM supplemented with 10% FBS and 1% AB. Twenty-four hours later ESCs were seeded onto the inactivated mouse embryonic fibroblasts and cultured in knockout DMEM (Life Technologies) supplemented with 15% ES-qualified FBS (Life Technologies), 0.1 mM nonessential amino acids (Sigma-Aldrich), 200 mM l-glutamine (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1% AB, and 500 U/ml leukaemia inhibitory factor (Chemicon, Billerica, MA, United States). Prior to transfection, ESCs were separated from mouse embryonic fibroblasts by the preplating technique and cultured on dishes coated with Matrigel Matrix Growth Factor Reduced (1 mg/ml DMEM; BD Biosciences, Becton-Dickinson, San Jose, CA, United States). The morphology of cells was analysed using a Nikon Eclipse TE200 microscope with Hoffman contrast. The cells were subjected either to Sdf-1 treatment, transfection with siRNA complementary to mRNA encoding CXCR4 or CXCR7, quantitative RT-PCR, immunolocalisation, or western blotting or used for co-culture experiments.

Cells seeded on 35-mm glass-bottom dishes (MatTek, Ashland, MA, USA) were incubated with 5 mg/ml Oregon Green 488 dextran 10 000 MW (Life Technologies) overnight, followed by 2 h chase in dextran-free medium. At the end of the chase period, the cells were rinsed with and maintained in the imaging buffer (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 10 mM HEPES, 10 mM Glucose, pH 7.4). Fluorescence images were acquired using an inverted Nikon ECLIPSE TE200 microscope at excitation wavelengths of 440 nm and 490 nm and emission wavelength of 545±50 nm, with a CCD camera controlled by InCytIM-2 software (Intracellular Imaging Inc., Cincinnati, OH, USA). The 490/440 ratio was calculated for each region of interest representing individual lysosomes or lysosomal clusters and converted into a pH value using the pH calibration curve generated from the same coverslip after the initial ratio measurement. To generate the lysosomal pH calibration curve, potassium isotonic solution (10 mM HEPES, 10 mM MES, 140 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM glucose) of pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7.0 containing 10 μg/ml nigericin were sequentially added to cells and each was equilibrated for 10 min before ratio images were taken. Lysosomal pH values were obtained by fitting the obtained intensity ratios to the standard curves.

Zeiss Axiovert 200 M microscope with Zeiss Apotome were used to collect immunofluorescent images. Bright-field images were collected using a Nikon Eclipse TE200 microscope. Data were analyzed using the Fiji package of ImageJ (v.1.0) and Photoshop screen overlay method (v. 21.1.0).

Control or IL-4-treated co-cultures of differentiating C2C12 myoblasts and either ESCs or MEFs or proliferating C2C12 myoblasts were fixed in cold methanol for 10 min at 4 °C at day 3, 6, or 9 of the co-culture. Next, cells were stained with Giemsa and May–Grunwald dyes according to the manufacturer’s protocol (Merck, Darmstadt, Germany) and examined with 20× objectives on Eclipse TE200 microscope (Nikon) in order to calculate the index of fusion. The index of fusion is the ratio of nuclei localized in multinucleated myotubes compared to the number of all observed nuclei. Images were taken with DXM 1200 digital camera and analyzed using NIS Elements F 2.30 software (Nikon, Tokyo, Japan). Ten representative fields of view of control and IL-4-treated co-cultures at each time point were analyzed.

A scratch assay within the myelinating co-culture was performed on either DIV 17 or DIV 24 according to [48 ], with a few modifications. To this end, a 20 µL sterile pipette tip was used to generate a scratch on the cell surface from the upper part of the cover slip vertically down to the lower part of the cover slip. During this procedure, constant speed, 60–80° angle and moderate pressure were kept equal to gain comparable scratches with a similar width on all coverslips. Directly afterward, the scratch medium was exchanged with the new medium additionally containing 0.02 µM parbendazole or DMSO as control. Afterward, pictures of the scratch were taken 3 days (t1 = 0 h, t2 = 24 h, t3 = 48 h) in a row until fixation. Pictures were taken using the Nikon eclipse TE200 microscope in 20× magnification at the upper and the lower edge of the scratch. In between the picture acquisition, cells were placed back into the incubator at 37 °C, 5% CO₂.