Lsm 510 axioplan 2 confocal microscope

Confocal microscopy was performed with Malme cells that were grown to sub-confluence (70 to 80 %) on acid-washed glass coverslips. After washing with PBS, cells were fixed and permeabilized with ice-cold methanol acetone solution (7:3) for 15 min at −20 °C. Cells were air dried and subsequently incubated with a blocking solution (0.5 % bovine serum albumin in PBS) for 1 h at room temperature. Subsequently, the cells were covered with 50 μl of the primary antibody anti-MICA (AMO1; 1 mg/ml) diluted in 1:50 blocking solution, and incubated overnight at 4 °C in a humidified atmosphere. After washing five times with PBS, 1 μl of the secondary Cy2-conjugated goat anti-mouse antibody (115-225-062, Jackson Laboratories, via Dianova, Hamburg, Germany) diluted in 100 μl blocking solution plus 1 μl of Hoechst 33342 (10 mg/ml) was added to the cells before incubation for 1 hour at room temperature. The coverslips were washed five times with PBS before they were mounted on microscope slides using fluorescence mounting medium (Dako, Hamburg, Germany). Microscopy was performed with the Zeiss LSM 510 Axioplan 2 confocal microscope.

For immunohistochemistry, 5-μm-thick paraffin sections of paraformaldehyde (PFA)-fixed kidneys were prepared. Before immunofluorescence staining, dewaxed sections were pretreated with blocking solution containing 5% normal goat serum in PBS with 0.1% bovine serum albumin and 0.3% Triton X-100 for 60 min. Without rinsing, sections were incubated with polyclonal rabbit anti-NCC (Millipore, 1:500) or anti-α-ENaC (Novus Biologicals, 1:500) antibodies at 4°C overnight. After washing, sections were incubated for 1.5 h with goat anti-rabbit Alexa 488 or goat anti-rabbit Alexa 568 secondary antibodies (Invitrogen, 1:400), respectively. Controls were performed by omitting primary antibodies. The slides were analyzed on a Zeiss LSM 510 Axioplan 2 confocal microscope equipped with a 63 × oil immersion lens (NA 1.3). Individual fluorochromes were simultaneously excited by lasers at 488- and 543-nm wavelengths with appropriate filter sets for the emitted light to avoid crosstalk. Images were merged using Adobe Photoshop.