Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(15)
Indirect Immunofluorescence Assay (IFA)
IFA and confocal microscopy were used to identify the neutrophil infiltrates in the squamous epithelia of cervico-vaginal sections of control, WHI-07-treated, and N-9-treat-ed mice. Cervicovaginal tissue sections were deparaffin-ized, hydrated through graded ethanol, and immunostained with a rat monoclonal antibody (mAb; Cederlane Laboratories Ltd., Westbury, NY) directed against mouse neutrophils (clone 5120-26.1-110). After antigen retrieval by heating (93 °C for 10 min in 10 mM sodium citrate buffer, pH 6.0), sections were preincubated with 10% normal goat serum in PBS and treated with optimal dilution (5 ^g/ml) of the mAb followed by FITC-conjugated goat anti-rat IgG (Pierce Chemical Co., Rockford, IL). Each incubation step lasted 60 min, with 5 min PBS washes between each step. The slides were counterstained with PI (1 ^g/ml), washed in deionized water, mounted in anti-fade (Bio-Rad), and visualized by laser confocal microscopy as described above. Negative controls were carried out by replacing the primary mAb with PBS or by using rat IgG2 as control mAb. buy asthma inhalers
Results are presented as mean or mean ± SD. Comparisons between WHI-01-, 05-, or 07-treated sperm and control sperm motility, acrosomal loss, and sperm binding to eggs were analyzed by a paired, two-tailed Student’s t-test. Linear regression analysis was used to find correlated values between two measured parameters, and t-test was used to find the significance of differences between two independent correlations. A p value of < 0.05 was considered significant. Nonlinear regression analysis was used to find IC50 and EC50 values from the concentration effect curves using GraphPad Software (San Diego, CA).