Fluoromount

Cells were grown in Millicell EZ chamber slides coated with poly-l-lysine and were further incubated with 100 µM BrdU for 2 h. Melanoma cells and control cells were fixed with 1:1 acetone solution for 30 min, DNA was hydrolyzed with 2N HCl for 30 min, and non-specific binding sites were blocked by 5% BSA dissolved in TBST. Melanoma cells and control cells were incubated overnight with 500 µL primary anti-BrdU antibody in TBST at a 1:10 dilution, washed thrice with TBST buffer. After that, cells were treated with 500 µL, Alexa Fluor conjugated 555 goat anti-mouse secondary antibody suspended in TBST at a 1:200 dilution in each well for 2 h at room temperature. Cell nuclei were stained with DAPI (1 µL/mL of DAPI in PBS) for 10 min, cells were fixed with formaldehyde and mounted with Fluoromount (Dako). Melanoma and control cells were examined under a fluorescence microscope at 40× magnification with AxioVision A5 microscope (Zeiss Inc., Oberkochen, Germany). BrdU-positive melanoma and control cells were counted manually from three independent images. The percentage of proliferating cells was assessed by calculating the number of BrdU-positive cells in the 3, 3′- (3, 5-DCPBC) treated group, and the DMSO treated control group in 5 to 6 random fields.

ChR2-GFP-V6.5 ESCs seeded on Menzel-glass coverslips were fixed by 4% paraformaldehyde for 30 min and washed three times with PBS (5 min per wash). Then, the cells were permeabilized with 0.3% Triton and blocked with 10% bovine serum albumin (BSA) at 37 °C for 60 min and then covered with primary antibody solution and transferred to a cold room at 4 °C overnight. The cells were washed three times, and secondary goat anti-mouse antibody conjugated to Alexa Fluor 555 (Molecular Probe, USA) was added for incubation at 37 °C for 60 min. After the cells were washed three times with PBS, 4′,6-diamidino-2-phenylindole (DAPI) was added for 10 min, and the cells were washed in water three times and mounted using Fluoromount (Dako, Denmark). The primary antibodies were mouse anti-OCT4 (Chemicon, 1:200), Rabbit-anti SOX2 (Abcam, 1:200), Rabbit-anti NANOG (Abcam, 1:200), Mouse-anti Ki67 (Abcam, 1:200) and mouse anti-SSEA1 (Chemicon, 1:200). The images were acquired with a Leica TCS SP2 inverted confocal microscopy system with an HCX PL APO CS 40× 1.25 NA oil immersion objective (Leica, USA). Immunostaining semi-quantification between non-light and light stimulation groups was done as previous [19 (link)].

For immunofluorescence, 7-μm cryosections were fixed in 4% paraformaldehyde, blocked for one hour in 10% goat serum in PBS and incubated for one hour at room temperature with specific antibodies against fibronectin (Sigma, St. Louis, MO, USA), collagen I (Chemicon, Temecula, CA, USA), F4/80 (Abcam, Cambridge, MA, USA), p-Smad-2 (Abcam, Cambridge, MA, USA), and dystrophin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). FITC-conjugated goat anti-rabbit IgG and rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. For monoclonal anti-mouse antibodies, all incubations were performed with mouse IgG-blocking solution from the MOM kit (Vector Lab, Burlingame, CA, USA) diluted in 0.01% Triton X-100/PBS. For nuclear staining, sections were incubated with 1 μg/ml Hoechst 33258 in PBS for ten minutes. After rinsing, the coverslips were mounted using Fluoromount (Dako, Carpinteria, CA, USA) and observed under a Nikon Diaphot inverted microscope equipped for epifluorescence [26 (link)].

Acute slice preparations were fixed in 4% PFA/4% sucrose (w/v, phosphate-buffered saline, PBS) at room temperature and stored at 4°C overnight in the same solution. After fixation, slices were washed in PBS and consecutively incubated for 1 hr with 10% (v/v) normal goat serum (NGS) in 0.5% (v/v) Triton X-100 containing PBS to reduce unspecific staining. For post hoc visualization of patched dentate granule cells, sections were incubated for 3 hr with streptavidin-Alexa Fluor 488 (Invitrogen, #S32354; 1:1000 dilution in 10% (v/v) NGS, 0.1% (v/v) Triton X-100 containing PBS) at room temperature. Sections were washed in PBS and incubated with DAPI for 10 min (Thermo Fisher Scientific, #62248; 1:5000 dilution in PBS) to visualize the cytoarchitecture. After washing, sections were transferred onto glass slides and mounted with fluorescence anti-fading mounting medium (DAKO Fluoromount). Confocal images were acquired using a Leica SP8 laser-scanning microscope equipped with a 20× multi-immersion (NA 0.75; Leica) and a 40× oil-immersion (NA 1.30; Leica) objective. Image stacks were acquired in tile scanning mode with the automated stitching function of the LasX software package.

After the last in vivo experiments mice were anesthetized by i.p. injection of ketamine (0.15 mL, 50 mg/mL). In total, 0.05 mL heparin was injected in the left hearth ventricle and the animal were intracardially perfused with 20–25 ml of phosphate buffer (0.1 M, pH 7.3, room temperature) and subsequently with 20–30 -ml of paraformaldehyde solution (PFA; 4% in 0.1 M PBS, pH 7.3, room temperature) both at 11 ml/min. The brain was extracted and postfixed in 4% PFA at 4 °C overnight. Afterward, it was rinsed three times with phosphate buffer and preserved in 30% sucrose (0.1 M phosphate buffer) at −20 °C until further processing. After unfreezing the brain was cut into 50-µm free-floating coronal slices with a microtome (Leica VT1000 S). Brain slices were mounted on microscope slides, embedded in Fluoromount (Dako), and covered by a glass cover. We acquired fluorescence stacks (3-µm z-steps) of R-CaMP1.07 expression with a confocal laser-scanning microscope (Olympus FV1000; 546-nm excitation wavelength).

Cells were seeded on coverslips coated with poly-l-Lysine in medium supplemented with 10% FBS. The cells were then pre-treated as indicated for 4 days. For BrdU staining, the cells were incubated with 5′-bromo-2′-deoxyuridine (BrdU, 50 µM; Sigma-Aldrich) for 2 h. Next, cells were fixed with 4% PFA followed by incubation in blocking solution. Primary anti-BrdU antibody was diluted in blocking solution. After washing, cells were incubated at room temperature with secondary antibodies diluted in blocking solution. Cell nuclei were stained using bisbenzimide (Hoechst 33258, 1 µg/ml; Sigma-Aldrich), and the cells were mounted in Fluoromount (Dako). Cells were examined under a fluorescence microscope at ×20 magnification with Olympus motorized inverted research microscope. The percentage of proliferating cells was estimated by calculating the number of BrdU-positive cells relative to the total, Hoechst 33258-stained, number of cells, from over 30 random fields.

The synaptosomes were attached to the polylysine-coated coverslips for 40 min at 4 °C, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min, permeabilized with 0.2% Triton X-100 for 60 min and incubated for 24 h with a mixture primary antibodies against vesicular transporter of glutamate type 1 (VGLUT1, 1:200; Abcam, Cambridge, UK) and GABA_A receptorα1 subunit(1:100; Abcam, Cambridge, UK) for 90 min at room temperature. After rinsing with blocking buffer, the synaptosomes were incubated with a mixture of goat anti-mouse DyLight 549-and goat anti-rabbit fluoresceinisothiocyanate (FITC)-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The synaptosomes were then washed three times with phosphate buffer and 0.1 M carbonate buffer (pH 9.2), and the coverslips were mounted with fluoromount (DAKO North America, Inc., Carpinteria, CA, USA). Images were acquired in an Image Xpress micro confocal (Molecular Devices, San Jose, CA, USA).