Figure 2

Confocal images of rat lung slice co-stained with either the LIVE^®or DEAD^®viability stain and the specific alveolar type I cell antibody, VIIIB2. A) A 200 μm rat lung slice incubated with the live stain (1 μM Calcein-AM) following live immunohistochemical staining with VIIIB2 primary antibody/Alexa Fluor 532 secondary antibody and excited at 488 nm (green emission) to show living cells. B) The same lung slice incubated with the live stain (1 μM Calcein-AM) following live immunohistochemical treatment with VIIIB2 primary antibody/Alexa Fluor 546 secondary antibody and excited at 532 nm (red emission) to show the Alexa Fluor 546 secondary antibody staining at alveolar type I cells. C) Overlay of images (A) and (B) showing that VIIIB2 immunoreactivity is clearly restricted to elongated, thin cells located at the edge of the alveolar space (indicated by the arrows), characteristic of ATI cells. D) A 200 μm rat lung slice incubated with the dead stain (4 μM EthD-1) following live immunohistochemical staining with VIIIB2 primary antibody/Alexa Fluor 488 secondary antibody and excited at 488 nm (green emission) to show secondary antibody staining of alveolar type I cells. E) The same lung slice incubated with the dead stain (4 μM EthD-1) following live immunohistochemical treatment with VIIIB2 primary antibody/Alexa Fluor 488 secondary antibody and excited at 532 nm (red emission) to show the EthD-1 positive, dead cell nuclei. F) Overlay of images (D) and (E) showing that VIIIB2 immunoreactivity and the dead cell stain do not co-localise. G) Graph showing quantification of dead cells per field (105 μM × 105 μM) following treatments indicated at the bottom of each data set.