The cell suspension was gently filtered through two layers of nylon gauze, layered on top of a linear gradient of 10-90% Per-coll (Pharmacia), and centrifuged at 150 X g for 10 min. Two distinct cell layers were formed. The lower layer, which contained more than 95% of viable cells with progressive motility, was diluted with three volumes of modified Tyrode’s solution and centrifuged at 150 X g to remove Percoll solution. The cells were resuspended in modified Tyrode’s solution (1.0 X 107 cells/ml) and used for the study. Throughout this study we used only the preparation in which more than 70% of cells were motile. buy flovent inhaler
Tissues were fixed in freshly prepared Bouin’s solution (0.2% picric acid and 2% formaldehyde in PBS) overnight, dehydrated in ethanol, infiltrated, and embedded in paraffin (Paraplast m.p. 56-57; Curtin Matheson Scientific Co., Houston, TX). Each tissue section (7 ^m) was mounted on a slide that had been precoated with a solution containing 0.25% gelatin (Gibco, Grand Island, NY) and 0.025% chromium potassium sulfate. Sections were dried at 45°C, de-paraffinized in xylene, and rehydrated through a gradient from alcohol to distilled water. The rehydrated sections were placed in a saturated lead thiocyanate solution and heated according to the method of von Wasielewski et al.. After the slides had been cooled at room temperature for 15 min, they were rinsed in distilled water and PBS each for 5 min.