Lsm 510 uv meta

The cells were seeded on a sterilized cover slips in 12-well plates at a concentration of 10⁵ cells per well. After reaching the 50% confluence of the monolayer, the culture media was replaced with full media containing the studied samples of the nanocomposites (siRNA-Au-LM1-P’ and siRNA-Au-LM2-P’). siRNA was obtained by annealing of oligoribonucleotides ORN2 and ORN3 (Table 2). Final concentration of siRNA during cell transfection procedure was 12.5 nM. The cells were incubated for 4 h at 37 °C in the CO₂ incubator. Then, the media was replaced with fresh one and cells were incubated for 24 h at 37 °C in the CO₂ incubator. After incubation, cells were washed three times with PBS and fixed with 4% paraformaldehyde in DMEM for 30 min, and washed twice with PBS. The cell-containing cover slips were mounted on the slides using ProLong Gold antifade reagent with DAPI (Life Technologies, Eugene, OR, USA). The slides were examined with confocal laser scanning microscope LSM 510 UV Meta (Carl Zeiss, Oberkochen, Germany). Three laser lines were used: 405 nm (to detect cell nuclei stained with DAPI), 488 nm (to detect GFP), and 633 nm (to detect Cy5.5 labeled siRNA).

PIMC (10⁴ cells per well) were seeded in 8-well Nunc Lab-Tek Chamber Slides (Invitrogen, Carlsbad, CA, USA). After reaching the 50% confluence cells were washed with IMDM, fixed with 3.7% paraformaldehyde in IMDM, and 0.2% Nonidet P-40 was used to permeabilize the cells. Slides were incubated for 2 hours at 37°С with primary antibodies against α-SMA (ab7817), aggrecan (ab36861), fibronectin (ab45688), СD34 (ab81289), VEGF-A (ab1316) (Abcam, Cambridge, UK), CD105 (M352701-2), CD31 (PECAM1) (M082301-2) (Dako, Agilent, Santa Clara, CA, USA), CD14 (2109035) (Sony Biotechnology Inc., San Jose, CA, USA), then incubated for 1 hour at 37°С with Alexa 488-conjugated secondary antibodies (ab150077 and ab1500117, Abcam, Cambridge, UK). Samples were stained with rhodamine phalloidin (Sigma-Aldrich, Saint Louis, MO, USA) for F-actin, and glass slides were covered with the Prolong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA) for nuclei staining, mounted with coverslips and analyzed using confocal laser scanning microscopes LSM 510 UV Meta or LSM 780 NLO (Carl Zeiss, Jena, Germany) using three laser lines at 405 nm (to detect cell nuclei stained with DAPI), 543 nm (to detect actin microfilaments stained with phalloidin-TRITC), and 488 nm (to detect Alexa Fluor 488 secondary antibodies).