Lsm710 laser scanning confocal microscopy

To measure mitochondrial membrane potentials, a 20- to 21-day-old adult fly was dissected in Schneider’s Drosophila medium and stained with 2.5 nM tetramethylrhodamine methyl ester (TMRM, Molecular Probes, MA) for 20 minutes. Then, it was washed 3 times with PBS for 10 minutes each and fixed in 4% PFA for 40 minutes. Finally, the samples were washed twice in PBS for 10 minutes each and mounted on a slide glass with SlowFade mounting solu. The slides were observed with LSM710 laser scanning confocal microscopy (Carl Zeiss, Germany) under 1,500× magnification.

The lung prepared as described above was instilled with 1 ml 4% paraformaldehyde in PBS(+) and fixed with 4% paraformaldehyde for 2 days at room temperature. The lung was embedded in 5% Seaplaque agarose (Lonza) or paraffin and then sectioned to 100 μm or 6 μm thickness, respectively, with a microslicer (DOSAKA EM). Paraffin sections were deparaffinized with xylene and ethanol and then rehydrated. The obtained sections were permeabilized with 0.5% TritonX-100 in PBS(+) for 1 h, stained with specific antibodies against HA (C102), FITC, ABCA3 (3C9), or Pro-SP-C (Abcam), followed by Alexa488-labeled or Alexa546-labeled secondary antibodies, and then mounted with ProLong Diamond reagent (Thermo Fisher Scientific). All images were analyzed using LSM710 laser scanning confocal microscopy (Zeiss).

HeLa cells were subcultured on coverslips in a 12-well tissue culture plate. Cells were washed once with PBS, fixed in 4% PFA for 15 minutes, and permeabilized with 0.5% PBST for 10 minutes. Then, the cells were washed with 0.1% PBST and incubated in blocking solution (3% BSA and 1% normal goat serum in PBST) for 1 hour. Primary antibodies were added to blocking solution (1:200) and the cells were incubated overnight at 4°C. After washing with PBST six times, cells were incubated with appropriate secondary antibodies (1:200) and Hoechst (33342, 1:2,000) in blocking solution for 1 hour at RT. The antibody-labeled cells were washed with PBS-T for six times and were mounted with mounting solution [100 mg/ml 1,4-diazabicyclo[2.2.2] octane (DABCO) in 90% glycerol]. The slides were observed with LSM710 laser scanning confocal microscopy (Carl Zeiss, Germany).