Dmi4000d microscope

Primary AEC isolated from lung biopsy were fixed in 3% paraformaldehyde (PFA) for 10 minutes at room temperature, washed three times in 0.1% glycin/PBS, and incubated with either rabbit polyclonal antibody to E-cadherin (1:200) (#ab53226) (Abcam, USA), or rabbit polyclonal SP-C (1:50) (#sc13979) (Santa Cruz Biotechnology, USA) or mouse monoclonal antibody to surfactant protein A (SP-A) (1:200) (#ab51891) (Abcam, USA) over night in a moist chamber at 4 °C. For detection Cy3 labelled anti rabbit secondary antibody was used for E-cadherin and SP-C, while a alkaline phosphatase labeling was used to detect SP-A. A549 cells were treated with TN or TG, for 18 hours and 7 days respectively and grown in either BMSC-cm or normal media for 24 hours. Cells were then fixed in 3% PFA as described above and subsequently stained overnight at 4 °C in a moist box with anti-cytokeratin, pan-FITC anti-mouse (1:200) (#C5992), mouse anti-vimentin-Cy3 (1:1000) (both: Sigma, USA) (#C9080) or caspase-3 (1:200) (#9662 S) (Cell Signaling, USA). Samples were then washed 3 times with PBS and analyzed under a confocal microscope LSM 510 Carl Zeiss or under Leica DMI4000D microscope.

Formalin-fixed human lung tissue sections were de-paraffinized in a xylene series and rehydrated through a decreasing ethanol series for immunohistochemistry. The slides were pre-treated by microwave in citrate buffer (100 mM, pH 7.0) for 10 minutes, washed 3x with 1x PBS and 0.1% tween (TBS). Slides were incubated overnight at 4 °C in an anti-Grp78 antibody dilution (1:100) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). EnVisionTM (DAKO, Bollschweil, Germany) was used for staining and detection, according to the manufacturer’s instructions. A Leica DMI 4000 D microscope was used to acquire images. For double immunofluorescence, tissue sections were incubated with primary antibodies specific to GRP78 (1:100) or SP-C (1:100) (surfactant protein C) (both: Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were anti-goat Cy3 labelled to detect GRP78, and anti-rabbit FITC labelled to detect SP-C (Abcam, USA); both secondary antibodies were diluted 1:1000.