Axioplan imager z2

The metaphases were arrested by incubation with Colcemid (#15210-040, KaryoMax Colcemid Solution (Invitrogen, Carlsbad, CA, USA) (10 ug/mL) 2 h prior to harvest. Cells were collected and treated with hypotonic solution (#685804, KCL 0.075M, Macron Chemical, Radnor, PA, USA) for 15 min at 37 °C and fixed with 3:1 ratio of methanol: acetic acid. Slides were prepared and aged overnight for use in G-band, SKY analysis. Chromosomes were stained with a trypsin- Giemsa staining technique [22 (link)]. Analyses were performed under an Axioplan ImagerZ2 (Zeiss, Dublin, CA, USA) microscope coupled with a CCD camera; images were captured with ASI software, (Applied Spectral Imaging Inc., Vista, CA, USA). The karyotype was determined by comparison to the standard ideogram of banding patterns for human chromosomes by ISCN 2009.

100 μl meiotic cell culture was added to 400 μl of 100% EtOH and stored at 4°C. For DNA staining, the cells were pelleted and resuspended in 20 μl of 1 μg/ml DAPI in PBS (13.7 mM NaCl, 270 μM KCl, 1 mM Na₂PO₄, 176 μM KH₂PO₄) and stored at 4°C. For visualization and scoring, 3 μl of sample on a 1-mm-thick glass slide (Fisher Scientific) and an 18 × 18 mm cover slip (VWR, Radnor, PA) was imaged on a Zeiss Axioplan Imager Z2 fluorescence microscope with a 100× Plan ApoChromat NA 1.45 oil lens. For each condition, 200 cells were scored.

100 μl of culture was added to 10 μl of 37% formaldehyde and incubated for 8–9 min at room temperature before pelleting. The supernatant was aspirated, 1 ml of 80% EtOH added, and tubes briefly vortexed. Cells were collected by short (~15 s) centrifugation, all traces of 80% EtOH removed by pipetting, the pellet was resuspended in 20 μl of 1 μg/ml DAPI in PBS, and stored at 4°C. For visualization, 3 μl of sample was transferred to a glass slide, a coverslip added, pressure applied to flatten the cells before viewing under a Zeiss Axioplan Imager Z2 fluorescence microscope with a 100× Plan ApoChromat NA 1.45 oil lens. All unbudded cells were scored for the presence of either one or two GFP dots (n = 100). All experiments were done in triplicate.