Alexa fluor 350 phalloidin

Unroofed cells were stained either with 16.5 pmol of Alexa Fluor 350-phalloidin (Life Technologies, A22281), Alexa Fluor 568-phalloidin (Life Technologies, A12380), or Alexa Fluor 647-phalloidin (Life Technologies, A22287) for 15 min depending on spectra of expressing FPs. Then cells were rinsed with PBS. 1 mm × 1 mm large montage was generated for proteins of interest and phalloidin using a Nikon Eclipse Ti inverted microscope with a 100×, 1.49 NA objective (Nikon, SR HP Apo TIRF) and an Andor iXon Ultra 897 EM-CCD camera under the control of Nikon Elements software. Images were obtained by TIRF illumination except for Alexa Fluor 350 which was imaged by epi illumination. This map was used to find the cells expressing the target proteins for FLIM and CLEM analysis. The imaged area was marked with a circle (4 mm in diameter) around the center of the imaged area using an objective diamond scriber (Leica, 11505059)^{40 (link),67 (link)}. The immersion oil was carefully removed from the bottom of the glass coverslip. The sample was subsequently imaged by FLIM or stored in 2% glutaraldehyde (Electron Microscopy Sciences, 16019) at 4 °C until EM sample preparation.

GECs were seeded at a density of 8 × 10⁴ on 17 mm glass coverslips (Warner Instruments) in four-well plates (Thermo Fisher Scientific). Cells were treated first with 3 mM ATP for 1 h and then infected with P. gingivalis for 3 and 24 h. The cells were fixed with 10% neutral buffered formalin, permeabilized by 0.1% Triton X-100 and blocked with 3% BSA for 45 min. The cells were stained for 1 h at room temperature with a mouse polyclonal antibody against Rac1 (1:500; Millipore) and a rabbit polyclonal antibody against P. gingivalis 33277 (1:1,000). The stained cells were washed and further incubated for 1 h at room temperature with Alexa Fluor 594 conjugated secondary goat anti-mouse polyclonal antibody and Alexa Fluor 488 conjugated secondary goat anti-rabbit polyclonal antibody (1:1,000; Invitrogen). The actin cytoskeleton was labeled using Alexa Fluor 350 phalloidin (1:500; Invitrogen). Coverslips with fixed cells were mounted onto Corning glass microscopy slides (Corning) using VectaShield mounting medium containing DAPI (Vector Laboratories). Images were acquired using Zeiss Axio Imager A1 epifluorescence microscope using QImaging MicroPublisher 3.3 cooled microscope camera and QCapture software.

PMA and PEA coverslips were coated with FN at 20 μg ml^–1 for 1 h. Human fibroblasts were seeded at a density of 5000 cells per cm² and incubated at 37 °C for 6 h and 22 h. Medium supplemented with 10% FBS was used. Samples were fixed with formaldehyde 4% at 4 °C for 30 min and next they were incubated with permeabilisation buffer for 5 min and blocked (1% v/v BSA/PBS) for 30 min. Next, samples were stained using primary monoclonal antibody against cellular FN (Abcam) for 1 h at room temperature. After they were washed (0.5% v/v Tween20/PBS), they were incubated with secondary Cy-3 anti-mouse antibody (Jackson ImmunoResearch) together with Alexa fluor 350 phalloidin (Invitrogen) for 1 h. Finally, samples were washed again and they were mounted using Vectashield without DAPI (Vector Laboratories, Inc.). Images were taken with an inverted fluorescent microscope (Zeiss AXIO Observer Z1) and image analysis was carried out using ImageJ.