Lsm 410

Ros 17/2.8 cells and MG63 cells were plated on chamber slides (NalgeNunc International, Naperville, IL, USA). When the cells were extended thoroughly, they were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 25 min. Following three washes with PBS, the cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Then they were blocked with 1% BSA for 1 h at room temperature followed by three washes with PBS. The cells were incubated with anti-actinin 4 antibody (Abcam, Cambridge, MA, USA) and anti-β₃ subunit antibody (Alomone, Jerusalem, Israel) at 4°C over night and 30 min at room temperature the next day with gently rocking. After washing with PBS for 10 min for three times, the cells were incubated with the secondary antibodies with labeled with Rhodamine or FITC (Jackson Lab., Barharbor, Maine, USA) at room temperature for 2.5 h shield from light. After three washes with PBS likewise, cells were counterstained with DAPI nuclear stain for 5 min and washed once with PBS, and mounted with Vectashield and reserved at 4°C shield from light. The fluorescent signals were visualized by confocal microscopy (LSM 410; Carl Zeiss, Oberkochen, Germany).The co-localization of actinin 4 and L-type calcium channel β₃ subunit was determined by evaluating at least five samples.

The pBI121-BplMYBs-GFP fusion gene and pBI121-GFP (control) were transiently expressed in onion epidermal cells using the particle bombardment (Bio-Rad) method. The transformed cells were then cultured on MS medium for 24–48 h and analyzed using a confocal laser-scanning microscope at 488 nm (LSM410, Zeiss, Jena, Germany).

HCT116 cells seeded on glass cover slips were stimulated with IL-6/sIL-6R for 72 h. After being washed twice with PBS, cells were fixed for 15 min at room temperature in paraformaldehyde (4% in PBS). Cells were then incubated with 0.1% Triton X-100 in PBS for 1 min for permeabilization. After being washed twice, cells were treated with 1% BSA in PBS for another 1 h. Cells were reacted with rabbit anti-E-cadherin antibody (Cell Signaling Technology, Danvers, MA, USA) (1:100 dilution in PBS) for 2 h at room temperature. Slides were washed twice and incubated with FITC-conjugated goat anti-rabbit IgG for another 1 h. DAPI containing mounting solution (SlowFad Gold, Thermo Fisher Scientific, Waltham, MA, USA) was used to mount the slides. E-cadherin distribution was then observed under a confocal microscope (Zeiss, LSM 410). Blue fluorescence (derived from DAPI) represented nuclei and green fluorescence indicated E-cadherin.

For direct observation of the effect of the drug on the dynamics of biofilm formation, Confocal Laser Scanning Microscopy was performed on bacterial biofilms of select organisms namely, S. aureus (ATCC 29213), S. epidermidis (ATCC 35984), and E. coli (ATCC 25922) with and without adding CurQDs as per need, on poly-L-lysine coated 8-well cell chambered slides (Nunc, Denmark) as described earlier with minor modifications (Pitts et al., 2003 (link); Schlafer and Meyer, 2016 (link)). Briefly, biofilms were formed in the presence of serially double-diluted concentrations of CurQDs at 37°C for 48 h. BHI-broth without the drug served as negative control. The biofilms were incubated with 6.6 μM concentration of DAPI (Invitrogen, United States) for 30 min in dark and were analyzed by Carl Zeiss Confocal systems.
The effect of drug CurQDs over 72 h bacterial biofilms was assessed as above. The chambers were visualized with the Zeiss LSM 410 with 63X 1.4 NA oil objective lens. All images were obtained and analyzed using ZenBlue imaging software. Co-localization maps were also constructed to study and interpret the interaction of autoflourescent Curcumin and DAPI.