Dispersed and compact chromatin demonstrated with a new EM method: phosphotungstic acid hematoxylin block-staining

Abstract

Glutaraldehyde/KMnO4 double fixation and phosphotungstic acid hematoxylin (PTAH) block-staining, before dehydration were found to reveal, with great detail and sharpness, the nuclear distribution of compact heterochromatin masses as electron-lucent patches. By contrast the areas of decondensed and dispersed chromatin acquired a high electron density due to the binding of the large PTAH molecule to basic groups in the loosened chromatin network. The method was tested on human blood leukocytes, on the thymus gland from immature rats, containing mitotic figures, and on mature avian erythrocytes. The results indicated that each cell type acquires a specific pattern of electron densities in the nucleus which depends upon the relative amounts of compact and dispersed chromatin present in that nucleus. Since the tissues are stained in-block immediately after fixation, artifacts of stain localization, due to alcohol dehydration, are avoided. Thus, PTAH block-staining "translates" the state of aggregation of the chromatin into characteristic and specific density patterns of the nuclei. This method may prove useful in differentiating active from inactive portions of the genome, at the ultrastructural level.