Slide preparation chromosome spreads

Table 3 lists the major steps involved in in situ hybridization of labeled probes to chromosomal targets in cultured cells. The first and second steps entail the preparation of chromosome spreads on slides. The third and... 

As the amount of fixative increases, the spreading of the nuclei and chromosomes improves, but the stainability of the chromosomes deteriorates. Gentle blowing on the slide also facilitates spreading and flattening of the preparation. 

Make the chromosome spreads. Under the stereo microscope in 1-2 drops (10-30 pL) of 45% acetic acid on a clean slide, tease the material to fragments with a fine needle, isolate the meristem, and remove all other tissue from the slides, in particular the root cap, which is tough and prevents squashing see Note 5). Apply a cover slip. Carefully tap the cover slip with a needle and then gently squash the material between glass slide and cover slip see Note 6). Check the preparation under a phase contrast microscope. 

Drop a volume of chromosome preparation on to a slide from a height and mark an area of spreads on the slide using a diamond pen. 

Air-dry the slides and store at - 70°C. If preparations dry too fast, chromosomes will not spread well (usually when the relative humidity is too low then use a humid chamber or ice-cold wet slides). 

Hypotonic pretreatment of seminiferous tubules can be done with distilled water. The swelling of cells encourages the spreading of chromosomes while preparing the slides. The duration of hypotonicity is varied depending upon the age of the animal. When young animals are to be studied, hypotonic treatment is less than 5 min, whereas seminiferous tubules from adults require at least 10 min of hypotonic treatment. Place hypotonically treated seminiferous tubules in 3 1 fixative (three parts of absolute ethyl alcohol and one part of glacial acetic acid) for 25 min. 

Note It is important that the dissecting needle be held vertical while tapping. When the dissecting needle hits the covershp, the tap compresses the stain and forces it away from the center of the preparation. As the dissecting needle moves away, the coverslip moves back up to its original position. The bouncing of the coverslip on the slide caused by the tapping sets up waves of stain that spread the chromosome arms away from each other. 

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