Chromosomes slide preparation

One of the most sensitive biological effects of ionizing radiation is to increase the frequency of normally observed chromosome aberrations (but not to induce qualitatively special abnormalities). Peripheral blood lymphocytes are the most feasible cells for chromosome investigations, as blood samples are easy to obtain and the techniques to stimulate the lymphocytes to proliferate within a culture medium and to prepare suitable chromosome slides for microscopic analyzation have their routine protocoil (e. g. Yunis, 1965 Lloyd et al, 1982). 

In essence the test screens mammalian red blood cells, which in their mature form lack a nucleus, for remnants of chromosomal fragments generated by mutagen exposure. These fragments are left behind when the nuclei are extruded from the maturing cells and they form the micronuclei for which the test is named. Experimentally, the test is normally conducted by administering the test substance by a route which enables it to enter the blood stream. In a t3T)ical assay four different dosages with five mice or rats per dose may be used. The bone marrow cells are isolated about 30 hours later and stained slides prepared. Approximately 1,000 red blood cells per animal are scored and the total test takes about 1 week to complete. 

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. 

The preparations most often affixed to silane- (9) or poly-L-lysine-coated slides are Carnoy-fixed suspensions of metaphase cells for chromosome studies (see Chapter 47) cytospins of cultured cells followed by methanol fixation sections of formalin-fixed, paraffin-embedded tissues and frozen sections fixed with acetone or ethanol/acetic acid after cutting. Each of these approaches imparts different intracellular effects that may modify cytological detail and/or hybridization. (See Chapters 8 and 9 for a discussion of fixatives and frozen-section preparations.)... 

In this test, cultured cells are seeded onto slides and the cells, which had been treated with and without metabolic activation for a short time period (e.g. 3 hours). Where negative or equivocal results are obtained, an independent experiment is conducted in which cells are treated for a long time period (e.g. 20 hours) in the absence of metabolic activation alone, and then sampled and examined for chromosome analysis. In both experiments the cells were sampled 20 hours after the start of treatment, as were the concurrent solvent and positive control cultures. Colcemide was added to each culture 2 hours before sampling in order to arrest cell division. Chromosome preparations were made, fixed, stained and examined. However, if clearly positive results were obtained in the first experiment, those from the second assay were not examined. If equivocal or negative results were... 

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... 

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). 

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. 

Treatment of slides with pepsin can increase probe reagent accessibility by digesting proteins. It is particularly effective if cytoplasm is associated with the chromosome preparations. Time and concentration of the pepsin treatment might need to be adjusted for different material. If the chromosome preparations are very clean, it can be left out (steps 4-6 of Section 3.1.). 

Different criteria have to be considered to guide the choice of a technique for preparing chromosomes of mammalian male germ cells. First of all, the method to be used will depend on the species to be studied and on the type of chromosome aberrations to be identified. Furthermore, certain methods are time consuming and/or involve the examination of many slides to get a large number of analyzable metaphases and to have a sample representative of the whole testis. In view of these considerations, the following methods are recommended ... 

Slides (2-3 days old) prepared according to Protocol 1.2. The most suitable chromosome preparations for in situ hybridization are those prepared according to Protocol 1.2 without colchicine pretreatment, but with hypotonic shock. 

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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