Axioplan 2 lsm510

At 16 h post-infection (pi), cells cultivated on glass coverslips were fixed with ice-cold methanol for 5 min, washed three times with PBS and incubated overnight in blocking solution (PBS, 0.01% NaN 3 , 1% BSA) at 4 • C. Coverslips were incubated for 2 h at room temperature with polyclonal mouse antibodies diluted 1/10 (anti-GroEL 1/100) in PBS, 0.1% saponin, 1% BSA. After three washing steps in PBS, 0.1% saponin, coverslips were incubated for 1 h at room temperature with a 1/500 dilution of AlexaFluor 488-conjugated goat anti-mouse (Invitrogen) and a 1/1000 dilution of DAPI (dilactate, D3571, Molecular Probes, OR, USA). After washing twice with PBS 0.1% saponin, once with PBS and once with deionized water, the coverslips were mounted onto glass slides using Mowiol (Sigma-Aldrich). Cells were observed under an epifluorescent microscope (Axioplan 2, Zeiss, Feldbach, Switzerland) and a confocal microscope (Axioplan 2 LSM 510, Zeiss).