Plan neofluar 40 1.3 na dic

For visualization of the α1 subunit of Na/K ATPase in DCs from jak3 -/-and jak3 +/+ mice, confocal microscopy was performed. Cultured DCs were seeded at 10 5 on Poly-L-Lysine (Sigma-Aldrich, Germany) coated chamber slides.
After 4 h, the attached cells were fixed with 4% PFA for 15 min at RT and permeabilized with 0.03% Triton-X100 for 10 min at RT. After blocking with 3% BSA in PBST cells were incubated at 4°C overnight with anti-ATP1A1antibody (1:100, Cell Signaling). The cells were rinsed three times with PBST and incubated with secondary goat anti-rabbit CF™ 488 antibody (1:300, Sigma-Aldrich, Germany) for 2h at room temperature. In addition cells were incubated for 30 min in the dark with DRAQ-5 dye (1:4000, Biostatus, Leicestershire, UK) for nuclei staining. After three washing steps, the slides were mounted with ProLong Gold antifade reagent (Life Technologies, USA). Images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC. Negative controls were carried out by incubation in absence of primary antibody.

For the visualization of eryptotic erythrocytes, 20 μl erythrocytes (1x10 6 cells) were fixed in methanol/ acetone and subsequently stained with Annexin V-FLUOS (1:100 dilution; Roche Diagnostics, Mannheim, Germany) in 200 μl Ringer solution containing 5 mM CaCl 2 . The erythrocytes were washed twice and finally resuspended in 200 μl Ringer solution containing 5 mM CaCl 2 . Forty μl were placed with PROlong Gold antifade reagent (Invitrogen, Darmstadt Germany) onto a glass slide, covered with a coverslip and images were taken on a Zeiss LSM 5 EXCITER confocal laser-scanning microscope or with the phase light (Carl Zeiss MicroImaging, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC. Scale bar 5 µm.