Prolong diamond with dapi

Human breast cancer MCF-7 cells (5 × 10⁴ cells per well; JCRB0134, JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan) were plated in 24-well plates in high-glucose DMEM with l-glutamine (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin at 37 °C in 5% CO₂. The culture medium was changed to Opti-MEM (Life Technologies, Carlsbad, CA, USA) 24 h after seeding. An additional 24 h later, cells were treated with Fc protein or mouse mNRG1-Fc with or without 10 mg ml⁻¹ heparin (Sigma-Aldrich) or neuropsin (10 mU ml⁻¹) in the culture medium for 60 min, washed and then incubated with an Alexa 488-conjugated donkey anti-rabbit IgG in fresh Opti-MEM (A21206; 1:1000, Molecular Probes, Eugene, OR, USA) for 1.5 h at room temperature. The cells were mounted in ProLong Diamond with DAPI (Life Technologies). Images were captured using a Zeiss LSM710 confocal laser-scanning microscope (Carl Zeiss MicroImaging, Jena, Germany). Fluorescence intensity was measured with ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

Nuclear fragmentation assays were performed as described previously^{23 (link),31 (link)}. In brief, CHO cells were plated on coverslips in 24-well dishes at a density of 2.5 × 10^{4 (link)} cells per well. After overnight incubation, cells were transfected with the plasmids indicated. Eighteen hours post-transfection, the cells were incubated with staurosporine (2 mg/ml) for 4 h at 37 °C and 5% CO₂. The cells were fixed with 4% paraformaldehyde (Alfa Aeser) in PBS (Biochrom) for 20 min at room temperature, permeabilized with ice-cold methanol for 30 s, quenched with 50 mM NH₄Cl (Roth) in PBS for 15 min at room temperature. The cells were mounted using ProLong Diamond with DAPI (ThermoFisher) to visualize the nucleus.

Adult mice were transcardially perfused with 20 mL of room temperature phosphate buffered saline (PBS, pH 7.4) followed by 20 mL of chilled 4% paraformaldehyde (pH 7.4). The brainstem was then removed from the animal and 150 um slices were made using a Zeiss VT100S vibratome. Slices were then incubated in a 0.5% Triton-X/PBS solution for 45 min to permeabilize the tissue. The slices remained in a 0.1% Triton-X/10% Fetal Bovine Serum (FBS, ThermoFisher)/PBS solution for a 12 hr primary antibody incubation of anti-mouse alpha smooth muscle actin (Sigma). The tissue was then washed three times in 0.1% Triton-X/10% FBS/PBS solution; the secondary antibody was incubated with the tissue after the third wash for 2 hr (donkey anti-mouse Alexa Fluor 647, ThermoFisher). The tissue was then washed three times in PBS before mounting on precleaned cover slides with Prolong Diamond with DAPI (ThermoFisher). Imaging of brain slices was achieved with a Leica SP8 confocal microscope.