Alexa fluor 555 phalloidin

Biocompatibility and cell morphology for the tested materials were studied using fluorescence (Olympus BX40 microscope, Olympus, Tokyo, Japan) and confocal microscopy (Carl Zeiss LSM780 Spectral Confocal, Carl Zeiss AG, Oberkochen, Germany). Cell viability was evaluated by acridine orange after 1 and 14 days of cell culture using fluorescence microscopy. The cells were stained for 1 min with 0.01% acridine orange solution, rinsed with PBS, and photographed.
The morphology of the cells was determined after 1 day of cell culture using confocal microscopy: Alexa Fluor^® 555 Phalloidin (Abcam, Cambridge, UK) and DAPI staining. SaOS-2 cultivated on membranes were fixed for 15 min with 4% paraformaldehyde, permeabilized for 10 min with 0.1% Triton X-100 in PBS, and then blocked for 20 min in 3% BSA in PBS. Alexa Fluor^® 555 Phalloidin (diluted 1:20 in PBS, Abcam, Cambridge, UK) was applied for 15 min, followed by rinsing with PBS and application of SlowFade mounting medium with DAPI.
All the data were expressed as means ± standard deviation (SD). Statistical analyses were performed by the one-way analysis of variance (one-way ANOVA). The statistical difference was considered statistically significant at p < 0.05. Statistically significant differences were indicated by lowercase letters.

The primary passage of cells in 12-well chambers was fixed in 4% paraformaldehyde for 30 min at room temperature. After that, Am-DCSCs were permeabilized in 0.3% Triton X-100 for 5 min and blocked in 5% BSA for 1 h. Then, Am-DCSCs were incubated with antibodies against STRO-1 (Novus Biologicals) at 1:150 dilution at 4 °C overnight. Alexa Fluor-648-conjugated anti-IgM (Yeasen) was used as a secondary antibody at 1:200 dilution, and DAPI (Abcam; 1:500) was used for nuclear counterstain. Bm-DCSCs and Am-DCSCs at P₃ were stained by Alexa Fluor 555 Phalloidin (Abcam; 1:200) and DAPI (Abcam; 1:500) as well for actin staining. Slides were examined with a confocal laser scanning microscope (CLSM; Leica).

Six rabbit anti-human-AR-V7 antibodies were compared in this study: clone EPR15656 (Abcam, VIC, Australia), clone E308L and “polyclonal antibody” (Cell Signalling, Danvers, MA, USA), clone SN8 (Creative Diagnostic, Shirley, NY, USA), clone DHH-1 (RQ4683, Assay Matrix, VIC, Australia), and clone RM7 (RevMab Biosciences, San Francisco, CA, USA), as well as the mouse anti-human-AR-V7 clone AG10008 (Precision Antibody, Columbia, MD, USA). The available information of antigens used for the anti-AR-V7 antibody generation is shown in Fig. 1A. Additional antibodies used in this study are: mouse anti-human AR-FL, clone ER179 (Abcam, NSW, Australia), rabbit anti-GAPDH clone 14C10 (Cell Signaling, VIC, Australia), Alexa fluor 488 goat anti-rabbit IgG (H + L) (LOT 1423009) or Alexa fluor 488 goat anti-Mouse (H + L) (LOT 1252783) (Life technologies, Eugene, OR, USA), horseradish peroxidase-labelled donkey anti-Rabbit IgG (1:1000 dilution) (Lot 9526417, GE Healthcare, Buckinghamshire, UK) or sheep anti-mouse IgG, Horseradish Peroxidase linked F(ab^’)₂ fragment (1:1000 dilution) (Lot 312511, Amersham, GE Healthcare, Buckinghamshire, UK) and Alexa fluor 555 Phalloidin (Abcam, NSW, Australia).