Silver Staining for Nucleolar Organizer Regions
Trude Schwarzacher and Molecular Cytogenetics Research Group
Borate buffer:0.01M Na2B4O7, pH=9.22
Aqueous silver nitrate solution:-
1 g AgN03 (reasonably high grade, clean, and not too old)
1 ml good distilled water
10 ul formalin (= .37% formaldehyde, optional, increases staining in old slides)
Mix well and put in a plastic syringe with a millipore filter.
Do not bring in contact with metal objects.
Silver nitrate stains clothes lab benches and fingers black, normally after about 10-1 6h. It is corrosive, but not really dangerous to human beings, and is used occasionally as a disinfectant. The skin sheds and the black spots disappear after a few days. It is advisable to wear gloves as the black spots are not very attractive. Cloths and lab benches are normally soiled for life.
Put a few drops of xylene on the preparation and cover with a large No.0 cover slip. Re-apply xylene if it dries out. For storage, leave the slide on the bench until all the xylene has evaporated, remove the coverslip carefully and put slide in a box. This method gives excellent contrast, but you have to cope with a little bit of xylene smell. Alternatively, you can examine the slides directly with immersion oil. This will not destroy the silver staining. Oil can be removed with xylene.
Nuclei should be visible without phase contrast. They should appear yellow to light brown and nucleoli brown. Too long staining results in black nucleoli and eventually black nuclei. If black precipitate occurs, slides were not clean or the silver nitrate solution was contaminated.
At metaphase, silver nitrate stains nucleolus organizer regions which have been active in the preceding interphase. Although amount of silver nitrate is in some relation to the amount of activity, or number of active, rDNA copies; it cannot be used for an accurate quantitative study, partly because of differences of silver staining strengths within and between slides depending on fixation procedures, density of nuclei, preparation quality and age of slides. Sometimes, e.g. with rye, heterochromatic regions also stain a pale brown color.
Kodama et al. 1980: An improved silver staining technique for nucleolus organizer regions by using nylon cloth. Jpn J Human Genet 25, 229-233
Schwarzacher T, Kraemer PM and Cram LS 1988: Spontaneous in vitro neoplastic evolution of cultures Chinese hamster cells. Nucleolus organizing region activity. Cancer Genet Cytogenet 35, 119-128.