Alexa flour 594 conjugated secondary antibody

Pulmonary fibroblasts were isolated from naïve mice as previously described in detail [34 (link)] and plated at 5 × 10⁵ cells/well in a 6-well tissue culture plate. Cocultures were created with the addition of MACS-isolated lung Gr-1⁺ myeloid cells (1 × 10⁵ cells/well) to the cultured fibroblasts for 24 h. Cell-free supernatants were collected to determine protein levels, and the remaining adherent cells in these cocultures were subjected to RNA isolation for quantitative PCR. Other cocultures were subjected to confocal microscopy to visualize collagen expression by the adherent fibroblasts. Briefly, adherent fibroblasts were fixed and permeabilized and incubated with an anti-collagen-1 mAb (Abcam) for 24 h followed by Alexa flour 594-conjugated secondary antibody (Life Technologies). Each slide was then treated with Prolog Gold Anti-Fade containing DAPI (Life Technologies), coverslips were mounted, and images were analyzed with a Zeiss LSM510 confocal microscope with C-Apochromatic 40x objective lens (Carl Zeiss). Images were assembled using Adobe Photoshop CS2 for Mac. In other experiments, cultured mouse fibroblasts were exposed to increasing concentrations of sTRAIL for 24 h and RNA was then isolated for quantitative PCR analysis.