The effects of staining several tissues with silver nitrate were studied in the electron microscope. Tissues were fixed in 2% glutaraldehyde buffered to pH 7.3 and containing 0.22 M sucrose, some being post-osmicated. Staining was effected by immersion of Araldite sections in unbuffered 5% silver nitrate for 30 min.
Increase in electron density was restricted to all nucleic acid-containing structures except mitochondrial DNA which is probably not associated with histones. Treatment of fixed tissues with cold 10% perchloric acid for 24 h, which extracts 85% of the total cell RNA, abolished silver staining within the nucleolus but did not affect that of cytoplasmic or mitochondrial ribosomes. Incubation of isolated, formalin-fixed liver cells with DNase I did not abolish nuclear staining. This evidence suggests that silver nitrate stains selectively the proteins associated with nucleic acids: the abolition of nucleoler staining by perchloric acid is not at present understood but may be due to some difference in relationship of protein to RNA in this, compared with other situations.
Silver staining indicates that the loci occupied by RNA-associated proteins differ in size and number in different type of ribosomes. Cytoplasmic ribosomes probably contain 5 loci of 4-6 nm diameter, 2 being situated in the 60-S subunit and 3 in the 40-S subunit. Mitochondrial ribosomes appear to contain 2 smaller loci. In the nucleolus the majority of the ribosomes constituting the granulosa contain 2 unequal loci of about 4 and 6 nm respectively, while the appearance of the pars fibrosa is compatible with the view that it consists of a network of long, extended RNA molecules with which small 2.5-nm protein loci are associated.