To verify staging, tissue samples were fixed in Bouin’s, embedded in polyester wax, sectioned at 6 ^m, affixed to gelatin-coated slides, and then baked at 40°C overnight. Sections were stained for 5 min with a fluorescent nucleo-philic dye, acridine orange (AO), to visualize distinctive patterns of cellular organization of spermatocysts at different stages. Images were viewed and recorded using an Olympus Fluoview Personal Confocal Microscope System (Olympus America Inc., Melville, NY) equipped with an Olympus IX70 inverted microscope and a UPLFL X10 objective (NA 0.30). The confocal system was controlled by the Fluoview 2.0.28 program for Windows NT. Illumination was provided by an argon/krypton laser at 488/568 nm and a 510-nm dichroic filter, a 550-nm long-pass emission filter, and a 6% neutral density filter. buy flovent inhaler
Statistical differences were examined by two-way AN-OVA, one-way ANOVA, or Student’s t-test (see figure legends; SigmaStat, Version 1.0, Jandel Scientific, San Rafael, CA). Where differences were detected, Student-Newman-Keuls multiple comparisons test was used to determine which values differed significantly (p < 0.05). In cases of non-normal distributions or unequal variances, log or square root transformations were performed.