Alexa fluor 488 labeled phalloidin

To examine cochlear whole mounts, surface preparations, and cryostat sections, tissues were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, as previously described [31 (link)]. Samples for surface preparations and cryostat sections were decalcified in 0.12 M ethylenediaminetetraacetic acid for 1 week at 4 °C or for 2 days at 23 °C. After permeabilization with phosphate-buffered saline containing 0.3% Triton X-100, the samples were incubated with Alexa Fluor 488-labeled phalloidin (Invitrogen) with/without DAPI for 1 h at 23 °C. The stained tissues were mounted in Prolong anti-fade (Invitrogen) with a coverslip and observed using an LSM700 confocal microscope.

Immunocytostaining was carried out as previously described.^{8 (link)} The peptide-chitosan matrices were prepared onto an eight-well chamber slide (Nalge Nunc), and HDFs (300 μL, 5 × 10³ cells/well) were incubated at 37°C for 90 min. HDFs were fixed and incubated with anti-vinculin antibody (hVIN-1), then stained with Rhodamine red-labeled secondary antibody, Alexa Fluor 488-labeled phalloidin (Invitrogen), and DAPI. The glass slides were examined under a FluoView FV1000D IX81 microscope (Olympus).

To examine the effects of the ZOL/β-TCP for osteoclast formation and activation, mBMSCs were cultured on each ZOL/β-TCP disc in accordance with the schedule of Figure 6. In brief, isolated mBMSCs (5.0 × 10⁵ cells/well) were cultured on ZOL/β-TCP discs in differentiation inducer medium, α-MEM with 10% FBS, 0.1% antibiotics, 30 ng/mL M-CSF and 50 ng/mL receptor for nuclear factor kappa B ligand (RANKL, Peprotech Inc., Cranbury, NJ, USA), for 5, 10 days at 37 °C. In this examination, we used the ZOL/β-TCP disc, immersing in culture medium for 24 h before seeding mBMSCs. The medium was exchanged every 2, 3 days. After culturing, to confirm osteoclast formation, cells were stained with Alexa Fluor^® 488-labeled phalloidin (Invitrogen, Carlsbad, CA, USA) for F-actin and DAPI (Dojindo, Kamimashiki, Kumamoto, Japan) for nuclei. Cells were washed with PBS (pH 7.3) and examined by observation with fluorescence microscopy (BZ-X710, Keyence, Osaka, Osaka, Japan). To confirm osteoclast activation, cells were performed with tartrate-resident acid phosphate (TRAP) staining using TRAP/ALP Stain Kit (Wako Pure Chemical, Osaka, Japan) and observed by phase microscopy (BZ-X710, Keyence, Osaka, Osaka, Japan). We analyzed three indexes from the results of observation: the osteoclasts area, cells size, and TRAP-positive area by BZ-X Analyzer (BZ-H3A (ver. 1.31), Keyence, Osaka, Osaka, Japan).

Panc1 cells were plated in 8-chamber slides and treated with fendiline for 24h and staining was carried out following the established protocols [70 (link)]. Secondary antibodies used included anti-rabbit Alexa Fluor 594 or anti-mouse Alexa Fluor 488. For staining of actin we used Alexa Fluor 488-labeled phalloidin (Invitrogen, Carlsbad, CA). Slides were mounted using Fuoro-Gel (Thermo Fisher Scientific). Zeiss Axioimager (Carl Zeiss Microimaging GmbH, Gottingen, Germany) was used to observe the slides and analysis was done using Axiovision Rel 4.8 software.

Suspensions of 2.0 × 10⁵ amoeba trophozoites were added to 4.0 × 10⁵ HRBC in a final volume of 150 μl M199s medium, distributed onto uncoated 35-mm glass-bottom culture dishes (MatTek 14 mm microwell, 1.5 cover glass, 0.16 to 0.19 mm), and incubated for 15 min at 37°C to allow amoebic phagocytosis. Nonadherent cells were aspirated off the plate, while adhered cells were fixed with 1.5 ml 4% formaldehyde for 20 min at room temperature (RT) and then permeabilized with 100 μl 0.2% Triton X-100 for 3 min. Cells were washed twice with 2 ml phosphate-buffered saline (PBS), blocked with 1.5% BSA-PBS for 30 min at RT, washed twice, and incubated for 1 h at RT with mouse monoclonal anti-HA antibody (clone HA-7; Sigma) (2.0 μg/ml) diluted in blocking buffer. After incubation, cells were washed with 2.0 ml PBS and stained for 1 h at RT with 100 μl goat anti-mouse IgG Alexa Fluor 568-conjugated antibody (Invitrogen) (10 μg/ml in blocking buffer). Cells were then washed and incubated for 1 h at RT with 100 μl Alexa Fluor 488-labeled phalloidin (Invitrogen) at 1:40 dilution in PBS. Cells were washed twice with 2.0 ml PBS, resuspended in 2.5 ml 0.02% sodium azide-PBS, and visualized using a 60× oil immersion lens objective (1.4 numerical aperture [NA]).

The BMSCs were seeded on both bare or blood pre-incubated collagen sponges and P-CS for 12 h (n = 6), as shown in Fig. 7D. The cells were fixed with 4% paraformaldehyde and permeabilized for in 0.15% Triton X-100. The cytoskeleton was stained with Alexa Fluor 488-labeled phalloidin (1:40, Invitrogen, Switzerland). All specimens were rinsed and mounted with Prolong Diamond Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen, ThermoFisher Scientific). The images were captured using confocal laser-scanning microscopy (Nikon A1R, Nikon Corporation, Minato-ku, Tokyo, Japan). Cell spreading area was calculated using Image J software.