Methods for the analysis of human chromosome aberrations / edited by K.E. Buckton, H.J. Evans.

Date:
1973
Catalogue details

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

Credit: Methods for the analysis of human chromosome aberrations / edited by K.E. Buckton, H.J. Evans. Source: Wellcome Collection.

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    Method 4. Fluorescence Analysis of Mitotic Chromosomes using Quinacrine Dihydrochloride Preparation of slides It is essential that the initial chromosome preparation should be of high quality, i.e., there should be minimal cytoplasm around the chromosomes, which should be well spread and not too contracted. For fixation a freshly prepared mixture of methanol and acetic acid (3:1) is used and the fixative is changed 3 times. Slides and cover-slips must be very clean. Staining 1. Stain slides for 6-8 minutes in 0.5% (w/v) quinacrine dihydrochloride in deionized water, pH 5.5-6.0, at room temperature (~20 °C). 2. Wash for 2-3 minutes in gently running tap water. 3. Rinse in deionized water. 4. Dry on a hot-plate (this step is optional). 5. Mount in deionized water. 6. Blot to remove the excess water; it is very important that only a thin layer of water should remain between the slide and cover-slip to reduce light scattering (flare). 7. Seal the edges of the cover-slip with rubber solution, etc. 8. Preferably examine the same day. If it is found that there is excessive flare, remove the cover-slip and remount. Microscopy The slides are examined under a high resolution fluorescent microscope with a vertical illuminator, in conjunction with an HBO 200 W/4 mercury lamp, a BG 12 exciter filter, and a 510 mm barrier filter. For a permanent record of the cell it is necessary to take photographs. Method 5. A Giemsa Staining Technique for the Identification of Human Chromosomes Preparation of slides 1. Ordinary newly made air-dried chromosome preparations are used. Much better results have been obtained with a hypotonic solution of potas sium chloride than with either sodium chloride or sodium citrate. 2. Incubate in 2xSSC for 1-16 hours at 60-65 °C.