Microscope cover glasses

Microscope coverglasses (Marienfeld GmbH & Co. KG, Lauda-Königshofen, Baden-Württemberg, Germany) with a diameter of 12 mm were coated with 10% poly-l-lysine (Santa Cruz) in water and washed three times with phosphate buffered saline (PBS) in a 24-well plate before cells were seeded onto them. The cells were fixed for 15 min in 4% paraformaldehyde in PBS. The fixated cells were washed two times with PBS and then immersed in 0.5% Triton X-100 in PBS for 3 min to permeabilize the cells, followed by three washes with PBS. The cells were blocked using PBS with 5% normal donkey serum (NDS) overnight at 4 °C, followed by incubation with rabbit anti-p65 (sc372, Santa Cruz) and mouse anti-β-tubulin (T8328, Sigma) at a 1:100 dilution in PBS with 5% NDS for 1 h at 37 °C. After three washes with 0.05% Tween-20 in PBS (PBST), the cells were incubated with secondary antibodies conjugated with different fluorophores at a 1:200 dilution in PBS with 5% NDS for 30 min at room temperature. Finally, the cells were washed three times with PBST, and then the cover glass was transferred onto a small drop of VECTASHIELD antifade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) on a microscope slide. The cells were examined with a confocal LSM (laser scanning microscope) 710 microscope (Carl Zeiss, Oberkochen, Baden Württemberg, Germany).