RESUMO
A simple technique is described for the routine in situ identification of the cellular composition of colonies and clusters in agar cultures of hemopoietic cells. The entire culture, dried and formalin vapor fixed within a Petri dish, is stained with a mixture of methylene blue and fast green. By this method cellular ribonucleoproteins (RNP), deoxyribonucleoproteins (DNP) and some cationic (arginine and lysine containing) proteins are detected. Different maturation stages of neutrophils, eosinophils, basophils, monocytes, and macrophages can be easily identified with colonies and clusters on the basis of the cytoplasmic and nuclear staining.
Assuntos
Células-Tronco Hematopoéticas/citologia , Azul de Metileno , Corantes de Rosanilina , Basófilos/ultraestrutura , Núcleo Celular/ultraestrutura , Células Cultivadas , Grânulos Citoplasmáticos/ultraestrutura , DNA/análise , Eosinófilos/ultraestrutura , Células-Tronco Hematopoéticas/análise , Células-Tronco Hematopoéticas/ultraestrutura , Histocitoquímica , Humanos , Macrófagos/ultraestrutura , Monócitos/ultraestrutura , Neutrófilos/ultraestrutura , Proteínas/análise , RNA/análise , Coloração e RotulagemRESUMO
A cytochemical method is suggested for the simultaneous and differential staining of cellular nucleoproteids [ribonucleoproteids (RNP) and desoxyribonucleoproteids (DNP)], as well as for the simultaneous contrast staining of some basic (arginine- and lysin-containing) proteins. The staining technique is based on DNA-denaturation procedures and the application of mixtures of basic dye--methylene blue and acid dyes--eosin or fast green at low concentrations. The combination of methylene blue with eosin is used for the staining of ribonucleoproteids (RNP) whereas methylene blue-fast green for the simultaneous detection of ribonucleoproteids and desoxyribonucleoproteids (RNP and DNP), as well as for the differential staining of nuclear DNP (after cold hydrolysis with 5 N HCl). The acid dyes eosin and fast green stain in pink resp. in green some cathionic proteins in the lysosomal (specific) granules of the neutrophilic and eosinophilic leucocytes in the cytoplasm of erythrocytes, and after cold hydrolysis in the cytoplasm of lymphocytes. A fluorescent variant of the method with sulfaflavin is also suggested for the fluorochromation of cytoplasmic cathionic granules in the luecocytes. Acid mucopolysaccharide components in the granules of basophilic leucocytes, tissue mastocytes and thrombocyres are stained intensively pink-violet (gamma-metachromatic). The possibilities for the application of the method in the quantitative analysis of blood and exfoliated cells, as well as for purpose of haematology, immunology and exfoliative cytology are discussed.
