Rhodamine conjugated phalloidin

RAW264.7 cells were incubated in a confocal culture dish (diameter, 35 mm) at adensity of 5 × 10⁴ cells·dish⁻¹ for 24 h, treated with 25 ng·mL⁻¹ RANKL and the screened compounds for five days, fixed with 4% paraformaldehyde for 30 min, stained with 5 μg·mL⁻¹ rhodamine-conjugated phalloidin (Sigma-Aldrich Chemical Co.) at 37 °C for 40 min, washed with PBS again, and stained again with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China) for 10 min at room temperature (Li et al., 2018). The F-actin ring of the osteoclasts was observed under a fluorescent microscope (Axiovert 200, Carl Zeiss, Oberkochen, Germany). The fluorescence images of the cells were processed using Zeiss ZEN software.

RAW 264.7 cells were seeded (3.0 × 10³ cells/well) on 96-well plates and treated or not with the indicated stimuli. After 5 days, the cells were fixed with 4% paraformaldehyde (PFA) for 20 min and F-actin ring-formation was evaluated by staining cells for 60 min at 37 °C with 10 µg/mL rhodamine-conjugated phalloidin (Sigma-Aldrich). Nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The samples were viewed by an epifluorescence microscope (Leica DMRB, Wetzlar, Germany) equipped with a digital camera.
The number of OCs in 40 µm² areas was counted in 9 random fields in triplicate and reported as F-actin rings positive cells containing 3 or more than 3 nuclei.

For fluorescence microscopy of live yeast to visualize GFP, cells were cultured in SR medium for 18 h at 30 °C, then, the appropriate amount of these cultures was suspended into fresh SG to reach an OD₆₀₀ of 0.3, and they were incubated for additional 4–6 h for GAL1 promoter induction. Cells were harvested by centrifugation and observed directly. To monitor vacuolar morphology and endocytosis, staining with FM4-64 was performed as described [21 (link)]. Nuclear labelling was performed by adding DAPI at 1:1000 directly to the harvested cells in vivo and washed once with PBS. To observe actin, yeast cells were fixed and treated with rhodamine-conjugated phalloidin (Sigma) as described [23 (link)].
Indirect immunofluorescence on yeast cells was performed as previously described [51 (link)]. Antibodies were used as follows: As primary antibody, monoclonal rat anti-alpha-tubulin (Serotec, YOL1/34) at 1:500 dilution; as secondary antibody, Alexa Fluor 594 anti-rat dye (Life Technologies) at 1:1000 dilution. DAPI was added at 1:1000 for nuclear labelling. Cells were examined in Eclipse TE2000U microscope (Nikon, Tokyo, Japan) and digital images were acquired with an Orca C4742-95-12ER charge-coupled-device camera (Hamamatsu Photonics, Hamamatsu City, Japan) and processed by HCImage and ImageJ software.