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Silver Staining for Nucleolar Organizer Regions Trude Schwarzacher and Molecular Cytogenetics Research Group Solutions: 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. Caution:- 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. Method:
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. Results: 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. References: 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. |