Perfusion-fixed testes to be used for sectioning were frozen in OCT compound (Fisher Scientific). Sections of 5-10-^m thickness were cut on a cryostat, collected on polylysine-coated slides, immediately treated with cold acetone (—20°C) for 5 min, and then air-dried. buy cheap antibiotics
Fixed testes to be used for collecting epithelial fragments were decapsulated, and then the seminiferous tubule masses were cut into small pieces with scalpel blades. The pieces were aspirated through 18-gauge and 21-gauge syringe needles and then sedimented at low speed in a clinical centrifuge. Fragments in the upper layer of sedimented material were collected with a pipette and attached to polylysine-coated slides. Excess fluid was removed, and the slides were immediately treated with cold acetone (— 20°C) for 5 min and air-dried.
Air-dried sections and epithelial fragments were rehydrated with TPBS (PBS, 0.05% Tween-20, 0.1% BSA) containing 5% normal rabbit serum (NRS) for 20 min at room temperature. After this, the material was incubated for 1 h at 37°C with either a 1:250 dilution of the dynein antibody or a 1:50 dilution of kinesin II antibody in TPBS containing 1% NRS. The sections were washed 3 times (10 min each wash) with TPBS and then incubated for 1 h with a 1:100 dilution of goat anti-mouse IgG conjugated to either fluorescein isothiocyanate or Texas Red. Sections were washed 3 times with TPBS and then mounted in Vectashield (Vector Labs., Burlingame, CA). In some cases, the second wash contained rhodamine or Oregon green phalloidin to stain for filamentous actin in junction plaques. Slides were observed and photographed on a Zeiss Axiophot microscope (Carl Zeiss, Inc., Thornwood, NJ) fitted with filter sets for detecting rhodamine and fluorescein. Negatives were scanned into digital format, and the images were manipulated using Photoshop 4 (Adobe Systems Incorporated, San Jose, CA) without altering the integrity of the data.