Vectashield containing 4 6 diamidino 2 phenylindole dapi

Human HER3+ breast cancer cells (SKBR3, BT474M1 and MDA-MB-468) were incubated with 1:100 HER3-VIA or GFP-VIA at 37 °C for 60 min (SKBR3, BT474M1) or 3 h (MDA-MB-468). After washing, fixation with 4% paraformaldehyde (PFA), and application of permeabilizing solution 2 (Becton Dickenson), nonspecific binding was blocked with 2.5% goat serum at 37 °C for 30 min. Cells were incubated with 1:100 Red™-conjugated anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories Inc. West Grove, PA, USA) in a dark chamber for 1 h at room temperature and washed with PBS. For the detection of EGFR, MDA-MB-468 cells were incubated with HER3-VIA for 3 h, then fixed and permeabilized. Cells were then labeled with cetuximab (40 μg/mL) for 10 min, followed by staining with Cy2-conjugated anti-human IgG antibody (ab97169, Abcam, Cambridge, MA, USA). For the detection of HER2, SKBR3 cells were labeled with trastuzumab (40 μg/mL) for 10 min, washed with medium, and then incubated with HER3-VIA for 3 h. Then, cells were fixed and permeabilized, and stained with Cy2-conjugated anti-human IgG antibody. Slides were mounted in VectaShield containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and images were acquired using a Zeiss Axio Observer wide-field fluorescence microscope (Carl Zeiss, München-Hallbergmoos, Germany).

After each time point, the globes were fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.2, for 30 minutes at room temperature (RT). Immunofluorescent staining was performed [17 (link)] by permeabilizing and blocking the tissue in blocking solution (PBS (93% v/v), Triton X-100 (10% v/v), BSA (5% v/v)) for one hour (hr) at RT. The tissue was incubated in primary antibody diluted in blocking solution overnight at 4°C (dilution will be described with each experiment) and washed with PBS. Alexa Fluor-conjugated 488 secondary antibodies (Invitrogen, Carlsbad, CA) were used at a dilution of 1 : 300 in blocking solution for 1 hr at RT prior to being washed again with PBS. The tissue was counter-stained with rhodamine-conjugated phalloidin (Invitrogen, 1 : 50) to visualize F-actin. Cells or tissues were mounted using VectaSHIELD containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA) to visualize cell nuclei. The primary antibody was excluded from the secondary control tissue. Following staining, the corneas were imaged using confocal microscopy.

For X-irradiation experiments, cells were plated on coverslips at a density of 20,000 cells/well and grown for 2 days. Due to practical reasons, samples were irradiated in T12.5 flasks for the carbon ion irradiation (vertical position). Irradiation with both radiation qualities was then performed with a series of doses as mentioned before. At various time points after irradiation (30 min, 1, 2, 4, 8, and 24 h), cells were fixed in 4% paraformaldehyde (Merck KGaA, Darmstadt, Germany) for at least 20 min at 4°C. Afterwards, cells were washed with PBS and permeabilized in 0.25% Triton (Sigma-Aldrich Co.) in PBS for 3 min. Subsequently, cells were probed with mouse anti-γ-H2AX antibody (ab26350, Abcam, Cambridge, UK) (1:300 dilution) and incubated overnight at 4°C. Next, the cells were washed with PBS and stained with Alexa Fluor 488 goat anti-mouse (H + L)-labeled antibody (A11001, Invitrogen, Life technologies) (1:300 dilution) for 2 h at room temperature. All antibody dilutions were prepared in 3% bovine serum albumin (BSA). Following this, three washing steps were performed with PBS after which a cover glass was mounted on the samples with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Brussels, Belgium).

Cultured OLs on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized for 5 min at room temperature in 0.1% Triton X-100 in PBS. The coverslips were blocked with Image-iT FX signal enhancer (blocking solution; Molecular Probes, Carlsbad, CA) for 1 hr and then incubated overnight at 4℃ with primary antibodies diluted in blocking solution. After rinsing, the cells were incubated with Alexa Fluor 488 - or 594-conjugated secondary antibodies for 1 hr at room temperature. Finally, the labeled coverslips were rinsed with PBS and mounted onto glass slides with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Images were captured with confocal microscopy (FV100D IX81; Olympus, Tokyo, Japan).