Eclipse ts100 inverted fluorescence microscope

To visualize the vascular-like network formation and macrophages in the co-culture immunocytochemical stainings were performed. The immunocytochemical staining was performed as described earlier (Huttala et al. 2015 (link)) except for the fixative used here was 4% paraformalaldehyde at RT for 20 min. After fixation, the cells were permeabilized with 0.5% Triton-X100 (MP Biochemicals, Ohio, USA) and non-specific binding sites were blocked with 10% BSA (Roche). Primary antibody dilution in 1% BSA in PBS was applied on the cells. Primary antibodies were α Anti-von Willebrant factor IgG (produced in rabbit, Sigma), CD11b and CD68 (both from BD). Secondary antibodies were also applied in 1% BSA in PBS solution. Secondary antibodies used were TRITC-labeled goat polyclonal antibody anti-rabbit IgG (Sigma), FITC-labeled goat polyclonal antibody anti-mouse IgG (Sigma), DAB Peroxidase (HRP) Substrate Kit, (Vector labs) and V450 (BD). After immunocytochemical staining the vascular-like network was photographed with Nikon Eclipse TS100 inverted fluorescence microscope (Nikon) and Nikon digital sight DS-U2 –camera (Nikon). Images were further processed with NIS Elements (Nikon) and Adobe Photoshop CS3-software (Adobe Systems Incorporated, San Jose, CA, United States).

Cells were seeded to 12-well plates (30,000 cells/well). Chaetocin and TRAIL simultaneous treatments (100 nM, 100 ng/ml, respectively) were performed for 6 h. Wells were rinsed once with PBS followed by incubation in 300 µl staining solution (1 µM YO-PRO-1 by Invitrogen Cat. No: Y3603, Thermo Fisher, USA and 1:1000 PI (1 mg/ml) in PBS) for 15 min at 37 °C in dark. Each well was visualized, and representative images were taken by Nikon Eclipse TS100 Inverted Fluorescence Microscope (Nikon Instruments Inc., NY, USA). Quantification of images was done with ImageJ software (NIH Image, NIH, Bethesda, USA).

Hematoxylin–eosin staining was performed by pipetting cell suspension containing macrophages on a microscopy glass and embedding them with Tissue-Tek^® O.C.T. Compound (Miles inc. Elkhart, IN, USA). Absolut alcohol, 94% ethanol, 70% ethanol and distilled water were added on the glass in sequence each for 1 min. Mayer hematoxylin was added for 10 min and this was rinsed with water and then with distilled water. 1% Eosin was added for 2 min followed by 96% ethanol, absolute ethanol and xylene. Cells were imaged with Nikon Eclipse TS100 inverted fluorescence microscope (Nikon, Tokyo, Japan) and Nikon digital sight DS-U2 –camera (Nikon).

For immunofluorescent microscopy of GS-5 10,000 to 12,000 cells were seeded on Laminin-coated 8-well chamber slides (Falcon, Corning, Amsterdam, NY, USA). Laminin-coating (10 µg/mL, sigma, L2020) was performed at 4 °C overnight. One day after seeding the cells were treated as indicated and fixed with 4% paraformaldehyde for 20 min at RT. The slides were washed with TBS-Tween (0.1%; TBS-T), blocked with 4% BSA in TBS with 0.3% Triton X-100 for 1 h at RT and primary antibody incubation occurred at 4 °C overnight. Hereafter, the slides were washed at least three times with TBS-T and secondary antibody was diluted 1:500 in TBS-T and incubated for 1 h at RT. After an additional wash with TBS-T the slides were mounted with DAPI containing Immunoselect antifading mounting medium (Dianova, Hamburg, Germany) or Fluoroshield with DAPI (Thermo Fisher, Frankfurt, Germany). Images were acquired with an Eclipse TS100 inverted fluorescence microscope (Nikon, Düsseldorf, Germany) operated by NIS Elements AR software (version 3.22, Nikon) or an AxioImager Z1 (Carl Zeiss, Jena, Germany).

BMM‐derived osteoclasts were cultured as described above in the absence or presence of increasing concentrations (1.5625, 3.125 and 6.25 μmol/L) of SO. When mature well‐spread osteoclasts were observed in RANKL‐only treated controls (around 6 days), cells were fixed with 4% PFA for 30 minutes, washed briefly with PBS and then permeabilized with 0.1% Triton X‐100 for 5 minutes at room temperature. Cells were then incubated with Actin‐Stain 488 Phalloidin for 30 minutes in the dark at room temperature. The nuclei were counterstained with 4ʹ,6‐diamidino‐2‐phenylindole (DAPI) for 5 mintues in the dark at room temperature. Fluorescence images were captured with the Eclipse TS100 Inverted Fluorescence Microscope (Nikon Instruments; Tokyo Japan). The number and average size of podosomal actin belt were quantified using ImageJ software.

For DNA damage analyses, 12,000 NCH644 cells/well were seeded on Laminin-coated 8-well chamber slides (Falcon, Corning, Amsterdam, NY, USA). Laminin-coating (10 µg/mL, Sigma-Aldrich, L2020) was performed at 4 °C overnight. One day after seeding, the cells were irradiated as indicated and, after an additional 24 h, were fixed with 4% paraformaldehyde for 20 min at RT. The slides were washed with TBS-Tween (0.1%; TBS-T), blocked with 4% BSA in TBS with 0.3% Triton X-100 for 1 h at RT, and the primary antibody incubation occurred at 4 °C overnight. Hereafter, the slides were washed at least three times with TBS-T, and a secondary antibody was diluted 1:500 in TBS-T and incubated for 1 h at RT. After an additional wash step with TBS-T, the slides were mounted with DAPI containing Immunoselect antifading mounting medium (Dianova, Hamburg, Germany) or Fluoroshield with DAPI (Thermo Fisher, Frankfurt, Germany). Images were acquired with an Eclipse TS100 inverted fluorescence microscope (Nikon, Düsseldorf, Germany) operated by NIS Elements AR software (version 3.22, Nikon).
The following primary and secondary antibodies were used: 53BP1 (NP-100-304, Bio-Techne GmbH, Wiesbaden, Germany), 1:1000; Alexa Fluor^® 488 F(ab’)2 fragment goat anti-rabbit IgG (H + L) (Thermo Fisher); 1:500.