| When checking your slides consider the following points:
- Scan slides carefully under phase contrast. Mark the general area on the slide where chromosomes are present underneath using a diamond pen: this may not be visible when placing hybridization solutions onto wet slides. Write down coordinates of good metaphase plates in order to find them again after in situ hybridization procedure. Look at the density of spreads, overall number of metaphases, how well spread they are, whether plates are complete, and how much cytoplasm is present. Also look for loss of material due to the removal of the coverslip: nuclei may have holes and metaphases may be incomplete (Fig. 8.1b). Make notes as these help to improve further chromosome preparation experiments and to select the best slides for the in situ hybridization experiment and may determine how to pretreat slides
- Possibly take photographs: chromosome morphology, and particularly centromeric constrictions, are normally much clearer than after the in situ hybridization procedure. However, not all cells will hybridize successfully
- Small chromosomes can be stained with DAPI (Protocol 9.3) and analyzed under the fluorescence microscope. After viewing, coverslips are removed: a) with immersion oil on the coverslip, warm slide to 37°C, gently but firmly pull off the coverslip being careful not to get oil on the slide surface and rinse the slide in 70% ethanol. b) without oil, help the coverslip to fall off in 70% ethanol. Transfer slides through a 70%, 90% and 96% ethanol series (2 min each) and air-dry
|
