Acti stain 488 fluorescent phalloidin

Actin staining with diamidino-2-phenylindole dihydrochloride (DAPI) counterstain was used to analyze changes in the structure of the cells’ cytoskeleton after particle exposure. For this purpose, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS; biochrom, Berlin, Germany). The following steps were performed protected from light and at room temperature. Before staining, cells were fixed with 4% paraformaldehyde (PFA; pH: 7.0). After 10 min, cells were rinsed with PBS for 30 seconds. Then, the cell membrane was permeabilized by adding a permeabilization buffer containing 0.05% Triton-X (Merck KGaA, Darmstadt, Germany) for 5 min. Osteoblasts were rewashed with PBS for 30 seconds before 100 nM actin staining solution (100 nM Acti-Stain 488 Fluorescent Phalloidin, Cytoskeleton, Denver, CO, USA) was added to the cells for 30 min. After washing three times with PBS, osteoblasts were incubated with DAPI (Merck KGaA, Darmstadt, Germany) for 5 min to counterstain the nuclei of the cells. The staining solution was removed, and cells were washed with PBS and stored at 4°C until microscopic examination.

Fluorescence microscopic analysis was performed to assess the actin filaments in PC3 cells without and with C12-HSL treatment using Acti-stain™ 488 fluorescent phalloidin (Cytoskeleton Inc., Denver, CO). Cells were cultured in chamber slides with DMSO vehicle (<0.01%) or with C12-HSL (50 μmol/L) in DMSO for 1 h, at 37°C and 5% CO2. Subsequently, the cells were then fixed with cold paraformaldehyde followed by addition of Triton-X 100 (0.1% in 1x PBS) to enhance membrane permeability. Cells were washed with phosphate buffered saline (PBS), labeled with phalloidin, and incubated for 1 h at RT. After washing with 1x PBS, cell nuclei were counterstained using 4′,6′-diamino-2-phenylindole (DAPI). The slides were visualized using a Nikon Eclipse E600 fluorescence microscope (Nikon Instruments Inc., Melville, NY).

dsRNA was fluorescently labelled using a Silencer siRNA Labelling Kit with Cy3 (Ambion, USA). Labelling of dsRNA was verified by decreased electrophoretic mobility compared with unlabelled dsRNA on an agarose gel. B. dorsalis midgut tissue from both the challenged and naïve groups were incubated with labelled dsRNA. For bafilomycin A1 treatment, midgut tissue was first incubated with 0.2 μM Baf for 30 minutes. Cy3-labelled dsRNA was then added to the reaction. The tissue was fixed for 20 min in 4% formaldehyde. Actin was visualized with Acti-stain™ 488 fluorescent phalloidin (Cytoskeleton, Inc., USA) following the instruction manual. Nuclei were counterstained with DAPI. Images were captured on an Olympus IX71 microscope driven by cellSens Dimension software (Olympus, Japan). All images were imported into and processed in Adobe Photoshop (Adobe, USA).