Plan apochromat 40 1.4 oil dic objective

Cells were seeded in 6-well plates (Sarstedt, Nümbrecht, Germany) with glass coverslips (VWR 631-0153, VWR, Gdańsk, Poland) at a density of 4 × 10⁴ cells/well. After 48 h treatment with Crataegus extracts (at concentrations close to the EC50 values), the cells were washed twice with PBS and incubated with 4% paraformaldehyde solution (PFA) at RT for 20 min. Then, the cells were washed with PBS, incubated with a 50 mM NH₄Cl solution for 30 min, and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Filamentous actin was visualized by staining with Alexa Fluor 488-conjugated phalloidin (1:40 in PBS) for 20 min. Next, the cells were washed with PBS/0.02% Triton X-100 and mounted by Vectashield anti-fade reagent containing DAPI to stain the nuclei. Microphotographs were collected using Zeiss LSM780, Inverted Axio Observer Z.1 with Plan Apochromat 40×/1.4 Oil DIC objective (Carl Zeiss AG, Jena, Germany). Images were prepared using the Zen Blue 2.1 software (Carl Zeiss Microscopy, Jena, Germany).

Cryosections were thawed to room temperature and after blocking with 2% BSA containing saponin, the sections were stained for specific antibodies at 4°C overnight. The sections were incubated with DyLight 488-conjugated secondary antibody for 2 h and nuclei stained with DAPI. A coverslip was mounted on the section with glycerol as the medium. Confocal images were taken on Zeiss LSM 710 Meta confocal laser scanning microscope (Carl Zeiss AG) using a plan-Apochromat 40×/1.4 Oil DIC objective (Carl Zeiss AG) and images were analyzed using ZEN 2009 software.