Axioscop 2 plus microscope

The cytological preparations were examined at the Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics, SB RAS, using an Axioscop 2 plus microscope (Zeiss) and objectives with various magnifications (Zeiss), and photographed using an AxioCam HRc camera (Zeiss); the images were analyzed using the AxioVision 4.7 microscopy software (Zeiss). Cell preparations were stained with fluorescent dyes and examined under an LSM 780 laser scanning confocal microscope (Zeiss) using the LSM Image Browser and ZEN 2010 software (Zeiss).

The testes of adult males were removed, separated from tunica albuginea and fixed in Bouin’s solution for 48 h, dehydrated in a graded ethanol series, immersed in xylene and finally embedded in paraffin. The testes were sectioned at a thickness of 10 μm and mounted on slides. The sections were deparaffinised, stained with hematoxylin and eosin and examined at low and high magnifications under an Axioscop 2 plus microscope (Carl Zeiss, Jena, Germany) equipped with a CCD camera AxioCam HRc (Carl Zeiss) and AxioVision image-processing package (Carl Zeiss). Only tubules of strictly frontal section were taken into the analysis. The stages of the seminiferous epithelium cycle were determined at the cross-sections of the testis according to Leblond and Clermont [24 (link)] and Wing and Christensen [25 (link)]. The ratios of various cell types of spermatogenic epithelium were estimated in the tubules at stages VI–VIII. Caudal epididymitis was minced in the phosphate buffered saline (PBS) at 37 °C. The sperm morphology was examined at the smear prepared on glass slides. Sperm abnormalities were classified according to Wyrobek and Bruce [26 (link)].

The pGEM-T-Easy-Pl-galectin-8 plasmid was used as template for the synthesis of Digoxygenin (DIG) labelled antisense and sense RNA probes. Whole-mount in situ hybridization was performed in a 96-well plate (Greiner Labortechnik, Longwood, FL, USA), using 30–40 embryos per well as previously described49 (link). The hybridization reactions were carried out at 62 °C, for at least 20 h. After hybridization, specimens were extensively washed and incubated with an anti-DIG alkaline phosphatase-conjugated antibody (Roche). Immuno-detection was carried out at room temperature with BCIP/NBT chromogenic substrates. Stained embryos were mounted on glass slides and observed under a Zeiss Axioscop 2 plus microscope. Images were captured by a digital camera. Control hybridization reactions with the sense probe did not show any specific signal.