Tcs sp5 scanner

Multi-label immunofluorescent staining with two or three rabbit antibodies was carried out using the tyramide signal amplification (TSA) kit (PerkinElmer) according to the manufacturers’ instructions. This allows for a very extensive dilution of the primary antibody, reducing cross-reactivity of the second secondary antibody to the first primary antibody to a minimum. Double-labeling was carried out with one TSA-reaction followed by normal immunofluorescent staining. Triple-labeling entailed two TSA-reactions followed by normal immunofluorescent staining. Secondary antibodies for TSA-reactions were swine anti-rabbit- HRP (Dako). For immunofluorescence donkey anti-rabbit conjugated to FITC or Cy3 (Jackson ImmunoResearch) or goat anti-rabbit Alexa Fluor 594 (Invitrogen) were used. Images were captured using a Leica DMI6000 CS confocal microscope connected to a Leica TCS SP5 scanner.

E. coli K12 MG1655 cells were tested for the ability to uptake extraneous RNA molecules. Selected RNA molecules (rrl_2585 and rrl_Pr) were synthesized and labeled with Cy5 on the 3′-ends (Syntol, Russia). Cells were cultured in 0.5 ml of LB medium at 30°C, 32°C or 37°C for 4 h, 8.5 h or 17 h with shaking in the presence of individual Cy5-labeled oligoribonucleotides added to a final concentration of 10 μM at the moment of inoculation. Thirty minutes before the end of culturing, MitoTracker™ Green FM (Thermo Fisher Scientific, USA) was added (2 μM) to provide for cell membrane staining. Following incubation, cells were harvested by centrifugation at room temperature (2,500 RPM) and twice washed with sterile phosphate buffered saline (PBS) to reduce the background fluorescence. After that, pelleted cells were resuspended in 20–50 μl of melted 0.8% agarose; 10 μl of suspension was placed on glass slides and pressed by coverslips. Fluorescent confocal microscopy imaging was obtained on a Leica DMI 6000 CS microscope (Leica, Germany) with a TCS SP5 scanner (Leica, Germany) and LAS X Software (Leica, Germany) with excitation/emission at 640/690 nm and 500/550 nm for Cy5 and MitoTracker™ Green FM, respectively.

HSC-T6 (1 × 10⁵) cells were seeded on slides containing poly-l-lysine coating and placed in a 6-well dish for overnight culture. Cells were treated with indicated concentrations of peach kernel extracts for 24 h. The cell medium was replaced with serum-free DMEM for 4 h, stimulated with 100 ng/mL LPS for 30 minutes, and then fixed with PBS containing 4% paraformaldehyde at 25°C for 2 h. Then, cells were perforated with PBS containing 0.1% Triton X-100 and incubated in a blocked buffer (PBS containing 2% BSA) for 1 h. Cells were stained with anti-Smad antibody followed by Alexa Fluor 488 anti-mouse IgG antibody (Invitrogen) for 1 h. Nuclei were stained with DAPI reagent for 15 minutes; then, slides were embedded with a mounting reagent and observed under a fluorescence microscope (Leica TCS SP5 scanner, Bensheim, Germany).