DNA cytochemistry in polytene chromosomes: electron contrasting agents for the ultrastructural detection of chromatin DNA after alkaline hydrolysis/methylation-acetylation.

Abstract

Salivary glands from Chironomus tentans larvae were fixed in glutaraldehyde and either subjected to alkaline hydrolysis followed by methylation-acetylation, or dehydrated without these treatments as controls. Ultrathin sections from Durcupan-embedded samples were contrasted by means of uranyl acetate, ruthenium red, indium trichloride, or the complex indium (III)-hematoxylin. Electron microscopic observations revealed a general contrasting pattern in control sections, while after the hydrolytic and blocking procedure only chromatin from polytene chromosomes appeared selectively contrasted. The nucleolus, Balbiani ring granules and puff materials showed weak or no electron opacity. After toluidine blue staining of semithin sections, an orthochromatic blue colour was found in chromatin bands from treated samples. These results indicate that alkaline hydrolysis/methylation-acetylation followed by contrasting with cationic heavy compounds is a valuable procedure to visualize chromatin DNA in polytene chromosomes.