Flu view 1000 confocal microscope

Log phase promastigotes, heat-shocked promastigotes and axenic amastigotes (1×10⁷ cells) were sedimented, washed twice with PBS and suspended in 1 ml of PBS. Aliquots of the suspension (2×10⁵ cells) were applied on microscopic slides. After fixing the cells for 2 min in ice-cold methanol, the slides were air-dried for 20 min. Non-adherent cells were removed by a gentle wash (0.1% Triton X-100 in PBS) followed by incubation in blocking solution (2% BSA, 0.1% Triton-X 100 in PBS). Slides were then incubated for 1 h with chicken anti-HSP23 antibody (1∶100 in blocking solution) and with either mouse anti-HSP70 (1∶125 in blocking solution), mouse anti-HSP90 (1∶250 in blocking solution) or mouse anti-HSP100 (1∶100 in blocking solution) antibodies. Cell were washed three times and than incubated for 1 h with anti-chicken-IgG antibody conjugated to Alexa Fluor 594 (Dianova, Hamburg, Germany, 1∶ 500), with anti-mouse-IgG antibody conjugated to FITC (Dianova, Hamburg, Germany, 1∶250) and DAPI (Sigma-Aldrich, München, Germany, 1∶25). After washing the slides three times, Mowiol and coverslips were applied and the slides were left to dry for 24 h at 4°C. Fluorescence microscopy was carried out on an Olympus FluView1000 confocal microscope (SIM-scanner and spectral detection).