Discrimination of the nucleolus by a critical electrolyte concentration method.

Abstract

A critical electrolyte concentration (CEC) method based on binding of toluidine blue molecules under Mg2+ competitive conditions has been proposed for discriminating differences in DNA-protein complexes in vitro (8) and associated with different states of chromatin supra-organization (1, 4). In the method proposed by Vidal and Mello (8), Mg2+ ions are added to the experimental staining solutions and the Mg2+ concentration at which metachromasy is prevented has been considered to be the CEC value.When studying variants of this technique, it was found that ribonucleoprotein complexes could be metachromatically highlighted after the CEC value of DNA-protein complexes was attained. This report describes a variant of the CEC technique in which the nucleolus is significantly discriminated. MATERIALS AND METHODSRoot tip cells of Allium ceps, salivary gland cells of Drosophila aracataca, Balb/3T3 cells, and NIH/3T3 cells transformed by transfection with DNA from carcinogen-treated human cells (5) were used.All the materials were fixed in an ethanol-acetic acid (3 : 1, v/v) mixture for 1 min, with the exception of the onion root tips, which lasted 15 min in the fixative. The glands of D, aracataca were squashed in a drop of 45% acetic acid after fixation, and frozen for removal of the coverslips, after which the preparations were rinsed in 70% ethanol and air dried.In the other materials, fixation was immediately followed by a 5-min bath in 70% ethanol. The onion root tips were subsequently processed for paraffin wax embedding and cut into 12-pm thick sections. The cultured cells were air dried immediately after the 70% ethanol treatment.Staining was performed with a 0.025% toluidine blue (Merck) solution in McIlvaine buffer at pH 4. 1, for 15 min. Immediately after staining, the preparations were treated with 0.02 M to 0.10 M aqueous MgC12 solutions for 15 min, rapidly rinsed in distilled water (5s), air dried, cleared in xylene and mounted in Canada balsam.Some preparations were treated with a 0.1 % RNase III (Sigma) aqueous solution at 37°C for 1 hr prior to staining.Spectral absorption curves were obtained for the nucleolus and the chromatin of the onion cell nuclei in order to detect their absorption peak wavelengths, and consequently, metachromasy loss at the chromatin level. Microspectrophotometry was carried out with Zeiss equipment (EMI 6256 photomultiplier, photometer 01, Planapo 100/1.25 objective, optovar 2, measuring diaphragm diameter of 0.

Índices Proliferativos Do Tumor Venéreo Canino Transmissível Pelas Técnicas Do Cec E Ki-67 Na Citologia Aspirativa Com Agulha Fina

“…As outras duas lâminas foram coradas para CEC com uma solução de 0,025% de azul de toluidina em tampão McIlvaine pH 4,0 seguida da aplicação de cloreto de magnésio (MELLO et al, 1993). Nestas lâminas fez-se a morfometria nuclear, sob observação em microscópio óptico adaptado ao analisador de imagem KS-300, sob objetiva de 100x, consistindo na determinação da área do núcleo e dos nucléolos, em 50 células tumorais escolhidas aleatoriamente, e a razão entre elas (área do núcleo : área do nucléolo).…”

“…Desta forma, a avaliação do número, forma e tamanho do nucléolo podem ser utilizados como parâmetro de avaliação proliferativa e de progressão neoplásica (BARBISAN et al, 1998). Por meio de técnicas citoquímicas usuais, como a da concentração eletrolítica crítica (CEC), podese obter imagens nucleolares nítidas, possibilitando sua análise morfológica e morfométrica (MELLO et al, 1993).…”

Morpho-ultrastructural alterations in the salivary glands of semi-engorged Riphicephalus sanguineus s.l. females subjected to permethrin

“…For the variant of the CEC [21], slides were stained with 0.025% Toluidine Blue in McIlvane's buffer (pH 4.0) for 20 minutes. After, the material was immersed in aqueous solution of 0.05M MgCl for 2, 5, 7, and 10 minutes to detect the ideal time at which metachromasy is abolished, with the removal of Toluidine Blue molecules bound to the chromatin by MgCl 2 .…”

“…Thus, the cation Mg +2 extracts the toluidine from the DNA ligation sites. Under these conditions, while the DNA metachromasy abolition occurs (green staining), the RNA remains metachromatic (blue or violet) [21].…”

“…The nucleolar activity is measured through the analysis of the nucleolus morphology (fibrillar and granular regions) and of the amount of RNA in the cytoplasm of the cell [21].…”