Hoechst 33258

For the immunofluorescence analyses, cells cultured on coverslips (Matsunami) were fixed with −20°C methanol for 7 min, or 4% PFA at room temperature for 30 min. Fixed cells were incubated with blocking buffer (1% bovine serum albumin in PBS containing 0.05% Triton X-100) for 30 min at room temperature. The cells were then incubated with primary antibodies (see below) in the blocking buffer for 1 h in a humid chamber. After washing with PBS, the cells were incubated with secondary antibodies and Hoechst 33258 (DOJINDO, 1:3000–1:5000) in the blocking buffer for 30 min, followed by a final wash with PBS. The coverslips were mounted onto glass slides (Matsunami) using ProLong Gold Antifade Mountant (Molecular Probes), with the cell side down. Images were acquired as z-stacks with an AxioImager M2 fluorescence microscope (Carl Zeiss) equipped with a 63×/1.4 or 40×/1.3 NA objective lens. The fluorescence intensity of centrosomal proteins was measured from the maximum projection images using FIJI (National Institutes of Health, Bethesda, MD, USA). The mean fluorescence intensity of a fixed-size area around the centrosome was calculated. The measurement was corrected for background intensity by subtracting the cytoplasmic signal within the same size area near the centrosome.

Calcein AM (Dojindo Molecular Technologies, Inc.) and Annexin V-Alexa Fluor® 647 (BioLegend) dyes were used to stain viable and apoptotic cells, respectively. Hoechst 33258 (Dojindo Molecular Technologies, Inc.) was used to stain the nuclei. The staining solution was prepared by mixing 10 µL Hoechst 33258 (1 mg mL⁻¹ stock concentration), 10 µL Calcein AM (1 mg mL⁻¹ stock concentration), 10 µL Annexin V (50 µg mL⁻¹ stock concentration), 500 µL Annexin V binding buffer (BioLegend), and 500 µL DMEM. The wash buffer comprised 500 µL of Annexin V binding buffer and 500 µL of DMEM. After treatment, the cells were washed twice with fresh DMEM, and 10 µL of staining solution was introduced into a cell culture chamber using a pipette via the adjacent inlet. The cells were then incubated at 37 °C for 30 min. Excess staining solution was removed by washing with 30 µL of wash buffer three times.

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline without calcium (PBS(−)), and permeabilized with 0.1% Triton X-100 in PBS, followed by pretreatment to block nonspecific reactions with 5% nonimmune goat serum in PBS(−). For collagen staining, the primary immunoreaction was carried out with a mouse monoclonal antibody against type-2 collagen (Abcam Inc., Cambridge, MA, USA). The secondary immunoreaction was carried out with Alexa 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) in 1% nonimmune goat serum in PBS, followed by rinsing with PBS(−). For cell nucleus staining, cells were incubated on 1 μg/ml Hoechst 33258 (Dojindo, Kumamoto, Japan) for 1 min, followed by rinsing with PBS(−). Fluorescent images were recorded with a fluorescence microscope.

The immunofluorescence staining of cultured cardiomyocytes was performed as previously described 11 (link), 46 (link). Briefly, the cardiomyocytes were cultured in a 24-well multiwall plate (Corning, Corning, New York, United States) and stained with monoclonal anti-α-actinin (sarcomeric) antibody (Sigma-Aldrich), and Alexa Fluor 555-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA). Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan) was used for nuclear staining. The surface area of the cardiomyocytes was automatically calculated using an ArrayScan system (Thermo Fisher Scientific) based on a positive indication for Alexa Fluor 555 (Thermo Fisher Scientific).

HeLa cells (1.5 × 10⁵ cells/well) were plated in 35-mm glass-bottomed dishes (3970-035; Iwaki, Japan) and cultured for 24 h. The cells were washed with DMEM supplemented with 2% FBS, and 1% Penicillin/ Streptomycin before incubation with the peptides in medium. After incubation for 3 h, the incubated medium was removed and the cells were washed three times with PBS. The cells were stained with 10 μg/mL Hoechst 33258 (Dojindo, Japan) for 15 min and subsequently were washed three times with PBS and twice with HBSS buffer for the next observation. The intracellular uptake of peptides was immediately observed, without fixation of the cells, using a confocal laser-scanning microscope (FV1000-D; Olympus, Japan).

Cells were fixed in 4% paraformaldehyde and immunostained with a rabbit anti-MICA polyclonal antibody (Abcam, Cambridge, UK), followed by the Alexa 488-conjugated anti-rabbit antibody (Life Technologies, Rockville, MD, USA) with Alexa 594-conjugated wheat germ agglutinin (Life Technologies), for labeling plasma membranes, and the nuclear dye Hoechst 33258 (Dojindo). Fluorescent images were obtained with Floid Cell Imaging Station (Life Technologies).

Transplanted tissues were fixed with 4% paraformaldehyde. Paraffin embedded tissue sections were stained with haematoxylin/eosin. Then, the paraffin sections were deparaffinized with xylene and sequential 100%, 95%, 80%, 70% ethanol treatments for 5 min each. The sections were treated with 10 mM citric acid (pH 6) at 95°C for 50 min followed by permeation with 0.4% Triton-X in PBS at room temperature for 30 min. The deparaffinized sections were stained with antibodies against human Lamin-A (1∶200; ab108595; Abcam), BEST1 (1∶200; ab2182; Abcam) and Ki-67 (1∶400; #9449; Cell Signaling). Nuclei were stained with Hoechst 33258 (Dojindo) and DAPI (Dojindo). hiPSC-derived RPE cells were collected in suspension and fixed with 4% paraformaldehyde followed by staining with antibodies against POU5F1 (OCT3/4) (1∶ 100; sc-5279; Santa Cruz), or BEST1 (1∶ 200; ab2182; Abcam). Antibodies were visualized with Alexa Fluor 488 goat anti-mouse (1∶ 1,000; Invitrogen) or Alexa Flour 488 goat anti-rabbit (1∶ 1,000; Invitrogen). Fluorescent microscopic images were captured with a fluorescent microscope (Olympus BX51, IX71, Tokyo, Japan).