Photometric sensys dp80 ccd camera

ND-FISH was performed as described by Fu et al. [28 (link)]. Briefly, air-dried, pre-treated slides were fixed for 10 min with 4% (w/v) paraformaldehyde, and then immersed in 2×saline sodium citrate (SSC) for 10 min. After dehydration in an ice-cold ethanol series of 75%, 95%, and 100% for 5 min each, they were air-dried These air-dried slides were then denatured at 80°C for 2 min in deionized formamide (60 μL per slide), followed by dehydration in 75%, 95%, and 100% alcohol at -20°C for 5 min each before air drying. A 10 μL aliquot of a hybridization mixture containing 0.5 μL FISH probe, 4.75 μL 2×SSC, and 4.75 μL 1×TE was applied to each slide, then the slides were incubated for 2 h at 37°C. The slides were counterstained with DAPI and Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). Sequential FISH analyses were performed as described in Fominaya et al. [35 (link)]. Digital images were captured using an Olympus BX-51 epifluorescence microscope equipped with a Photometric SenSys Olympus DP80 CCD camera (Olympus, Tokyo), and processed using Photoshop V7.0 (Adobe Systems Incorporated, San Jose, CA). After capturing each image, the slides were washed as described by Komuro et al. [37 (link)]. To make results reliable, at least 3 slides for each accession were performed FISH analysis, and for each slide, images for at least 30 cells were captured.