The polymorphonuclear cell population

Tuesday, October 14th, 2014 No Commented
Categorized Under: Medicine

Nonspecific labeling was blocked by coating with normal goat serum (X0907 [diluted 1:20]; DAKO) for 20 min at room temperature. The following primary antibodies were used: for HO-1 staining, the anti-HO-1 rabbit polyclonal antihuman antibody (sc10789 [diluted 1:50]; Santa Cruz Biotechnology, Inc; Santa Cruz, CA) overnight at 4°C; for iNOS staining, the anti-iNOS rabbit polyclonal antihuman antibody (sc8310 [diluted 1:50]; Santa Cruz Biotechnology, Inc) overnight at 4°C; and for nitrotyrosine staining, the antinitrotyrosine rabbit antihuman antibody (diluted 1:100) [Upstate; Lake Placid, NY] for 1 h at room temperature.

After being incubated with the primary antibody Cheap Tadalafil Australia, the slides were washed, followed by staining with the secondary antibody-alkaline phosphatase anti-alkaline phosphatase complex for 30 min at room temperature. After washing, the slides were stained with Fast Red for 15 min at room temperature. Dilutions and washing steps were performed with the preincubation solution. Slides were counterstained with hematoxylin for 5 min and mounted (Glycergel, C0563; DAKO). Negative control experiments for nonspecific binding were performed in a similar manner but in the absence of the primary antibody. The primary antibody was replaced by nonspecific Ig of the same species as the primary antibody or the preincubation solution. On immunostained slides, polymorphonuclear cells and macrophages were identified by morphologic analysis, and positively stained cells within 300 cells of each population were counted using a light microscope. Results were expressed as the percentage of positive cells within each leukocyte population.

Within the polymorphonuclear cell population, there were mostly neutrophils and very few eosinophils, as was shown on slides stained with May-Grunwald-Giemsa stain (Table 2). However, to be precise we use the term polymorphonuclear cells for the entire analysis of the study. All analyses were performed in a blind fashion by two investigators, and the results were averaged. Three replicate measurements were performed by each observer in 10 randomly selected slides. Both intraobserver and interobserver coefficient of variation (CV) were < 10%.