Human chromosome methodology / edited by Jorge J. Yunis.

Date:
1965
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Human chromosome methodology / edited by Jorge J. Yunis. Source: Wellcome Collection.

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    Autoradiography of Human Chromosomes 101 of aceto orcein stain (2%) are added as corresponds to the volume of the cell pellet (usually 2 to 3 drops). With a capillary pipet, cells and stain are mixed. The cells are then ready for squash preparations. The slides to be used are alcohol-cleaned, but the cover glasses are simply wiped clean with a lint-free cloth. If this is done, more cells will adhere to the slide when, later in the procedure, the cover slips are removed. There are certain brands of cover glasses that have to be siliconized. If the cell suspension contains large particles or debris which prevent a complete flattening of the cells, a drop can first be placed on a slide where these particles will attach to the glass. Then the remaining fluid is permit¬ ted to flow to the surface of another slide where it will be squashed. A filter paper is placed over the cover slip; squashing is then begun very gently by using a thumb. When stain can no longer be pressed out from under the cover glass, a final strong pressure is applied. Every sideways movement of the cover glass will destroy the preparation. Air pockets under the cover glass should be avoided because, later in the freezing procedure, cells near the air pockets will be dislodged and lost. Microscopic inspection of the slide at this stage will reveal whether the preparation has the necessary high quality to be of use in an autoradio¬ graphic study. If so, the slide is immediately sealed with Kroenig's wax along the edges of the cover slip to prevent desiccation. When not in use, the temporary slides should be stored in a refrigerator. We found that slides kept at room temperature for a period of time show a high background in the eventual autoradiographs. It is possible that some DNA becomes decomposed, thus allowing the label to difl^use into the cytoplasm. At 4°С the temporary slides can be stored safely for at least 4 weeks. From each culture, several good slides are generally used for observation and photography ; from each slide, the best mitotic figures, preferably those without overlapping chromosomes, are chosen. A total of about 30 photographed figures is necessary for most studies. The reason for scattering suitable cells over several slides is to avoid severe loss in case of accidents that can happen through a variety of manipulations for autoradiography. The coordinates of each cell in relation to the microscope stage are recorded to allow future relocation of the same. As soon as a sufficient number of cells have been photographed, the preparations must be processed further, i.e., the acetic acid must be removed and the slides air-dried. For this purpose, a freezing procedure (Conger and Fairchild, 1953) is applied. The slides are placed, right side up, on the smooth surface of a block of dry ice, where they are left for approxi- \