Understanding Romanowsky staining

Summary Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.     

Patterns of silver staining on NORs of prematurely condensed muntjac chromosomes following RNA inhibition

Prematurely condensed chromosomes of muntjac G0 lymphocytes as well as contact-inhibited and Actinomycin D (actD)-treated fibroblasts have been stained with silver nitrate to estimate the correlation between RNA suppression and the NOR staining. The results demonstrate that actD treatment for up to 36 h does not significantly affect the staining. Only partial suppression occurs in contact-inhibited cells, whereas complete abolition is obtained in long quiescent lymphocytes. We conclude that the reduction of the staining occurs only gradually from the NORs over a number of days or even weeks. We assume that the silver staining proteins may be associated with rDNA having a regulatory or structural role to play in rDNA activity.     

Automated Feulgen staining with a temperature-controlled staining machine

A Shandon Varistain 24-3 staining machine was modified in order to run automated DNA Feulgen staining. Initial studies showed a strict dependence of the staining intensity (integrated optical density [IOD]) on the temperature of the DNA hydrolysis in 4 N HCl: a difference of 0.5 degrees C around the optimum hydrolysis temperature of 27.5 degrees C resulted in IOD differences of up to 7.8% in epithelial cells and up to 12.0% in lymphocytes. A temperature-controlled stainless steel cuvette, covered with a 4 N HCl-resistant material, was developed and integrated into the machine. Temperature measurements were performed at different positions in the cuvette and on glass slides with copper-constantan electrodes fixed on them; no temperature gradient could be detected within the cuvette. The adjusted temperature of 27.5 degrees C remained constant over 24 hours. The coefficient of variation (CV) of the staining intensity in lymphocytes between different areas on the same slide and between different slides of the same staining cycle was less than 0.6%. The CV between different staining cycles was 5.9%. This system for automated Feulgen staining thus gives reproducible and reliable results and may be introduced into routine diagnostic procedures.     

Orcein staining of leukocytes

An Orcein staining method has been developed which stains mature and immature leukocytes in blood films and bone-marrow smears. Two different patterns of staining are obtained depending upon whether staining is or is not preceded by oxidation. In the latter case, all granulocytes and some monocytes show granular reddish-brown cytoplasmic staining. When prior oxidation is used, the staining is in the form of fine grey or black cytoplasmic granules. All lymphocytes, by both techniques, are negative. It is suggested that Orcein stains sulphated mucosubstances, possibly chondroitin sulphate, which in granulocytes is concentrated in their primary granules.     

Biochemical and immunohistochemical studies on overgrown gingival tissues associated with mannosidosis.

The gingival tissues of a male patient suffering from mannosidosis and presenting with gingival overgrowth have been studied. Routine histological assessment highlighted the presence of highly enlarged and vacuolated lymphocytes. The morphology of the connective tissues, fibroblasts and epithelium appeared normal. Immunohistochemical staining of the tissues for chondroitin sulfate proteoglycan demonstrated a normal distribution of this component throughout the connective tissues and intense staining associated with the vacuolated lymphocytes. In vitro studies indicated that fibroblasts isolated from the overgrown tissue did not differ from age and sex matched control fibroblasts with respect to proliferation, protein and proteoglycan synthesis. Taken together, these findings imply that the gingvial overgrowth noted in this patient was not due to a defect in the resident fibroblasts but rather reflected a secondary response to the tissues to impaired host defence mechanisms.     

Intercellular NOR-Ag-variability in man. II. Search for determining factors,clonal analysis

Summary Intercellular, nonartifactual variability of nucleolar organizer region (NOR)-Ag-staining was studied in cultured human peripheral blood lymphocytes, skin and embryonic fibroblasts. No differences in number and character of variable NORs and intensity of their staining were observed between lymphocytes stimulated to proliferate with phytohemagglutinin and pokeweed mitogen, as well as lymphocytes of first- and second division. The number of NOR associations per cell and the number of associated chromosomes per association were also similar. In a given individual these criteria were similar in lymphocytes and fibroblasts.In all nine clones derived from three independent parental fibroblast cultures the intercellular NOR-Ag-variability was similar to that observed in a given parental cell line. A significant decrease in the number of metaphases containing NOR associations was observed in second-division lymphocytes compared with first-division ones, as well as in skin fibroblasts compared with lymphocytes.     

Nile red staining of lysosomal phospholipid inclusions.

We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.     

Immunofluorescent staining of keratin fibers in cultured cells.

Antibody prepared against a group of keratins purified from human stratum corneum was used to identify cells containing keratins by immunofluorescence. In sectioned tissue and in culture, keratinocytes of skin and other stratified squamous epithelia-whether human, rabbit of mouse-stained strongly, indicating homologous amino acid sequences in the keratins of these species. In all cases, the antibody revealed a dense cytoplasmic network of discrete fibers probably consisting of aggregated (tono-) filaments. The pattern of staining was not affected by cytochalasin B or colcemid. No keratins were detected in cultured cells of mesenchymal origin (3T3, NIL, BHK, human diploid fibroblasts) or in connective tissues, indicating that the 100 A filaments of fibroblasts are not related to the keratins. Keratinocytes at all stages of differentiation, including basal cells, stained brightly and therefore contained abundant keratins.     

人胚胎干细胞传代培养及细胞化学染色特性

目的 体外建立人胚胎干细胞传代培养方法,研究人胚胎干细胞细胞化学染色特性.方法 以小鼠胚胎成纤维细胞作为饲养层传代培养人胚胎干细胞,检测人胚胎干细胞、自发分化克隆及拟胚体的细胞化学染色特性.结果 人胚胎干细胞在小鼠胚胎成纤维细胞饲养层上传30代以上其形态保持不变;人胚胎十细胞碱性磷酸酶、过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性,自发分化克隆细胞阳性程度明显减弱;人胚胎干细胞形成的拟胚体碱性磷酸酶染色弱阳性,过碘酸-雪夫反应、α-醋酸萘酚酯酶染色阳性.结论 小鼠胚胎成纤维细胞能支持人胚胎干细胞传代培养,细胞化学染色结果能初步鉴别人胚胎干细胞未分化特性.     

A silver staining method for single-cell gel assay.

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183-1186, 2001)     

Surface coats on human lymphocytes: freeze-drying and staining with cations.

Surface coats can be demonstrated on human peripheral blood lymphocytes by staining with ruthenium red, alcian blue, Thorotrast, and cationized ferritin, which are similar in distribution to a 40- to 65-nm layer of amorphous extracellular material recently reported on fixed, freeze-dried lymphocytes. Several additional lines of evidence, including X-ray microanalysis, suggest that the latter is not a contaminant added by freeze-drying. Freeze-drying may provide the means for a morphological assessment of the lymphocyte surface, including the extracellular coat, which may give additional insight into the immune response.     

Fluid hexagonal lattices of sub-units in negative staining preparations of fragmented human lymphocytes.

Electron microscopic studies of negative staining preparations of fragmented human lymphocytes have revealed highly-ordered fluid hexagonal lattices of repeating 100 A sub-units with central depressions. It is supposed that the particles came from the plasma membrane of the lymphocytes.     

Adhesion and transcellular migration of neutrophils and B lymphocytes on fibroblasts

During tissue inflammation, infiltrated leukocytes may have physical contacts with fibroblasts. We observed that neutrophils and B lymphocytes adhered in a larger proportion than T cells on cultured fibroblasts. Microscopy showed that adhesion was also characterized by leukocyte engulfment by the fibroblasts. In migration assays, only neutrophils and B lymphocytes were selectively able to migrate through a fibroblast barrier. Adhesion and migration were increased by stimulation with tumor necrosis factor-α (TNF-α) and phorbol-12-myristate-13-acetate (PMA). Antibodies against ICAM-1/β2 integrin blocked the interaction of neutrophils to fibroblasts. For B lymphocytes the couple VCAM-1/α4 integrin was also involved in this interaction. Human skin fibroblasts presented similar adhesion characteristics as rat cardiac fibroblasts. By measuring the distance between the border of migration holes and cadherin-positive adherens junctions, more than 65% of the holes correspond to the transcellular route over the paracellular route. Furthermore, vimentin staining revealed that the migration holes were highly nested by intermediate filaments in accordance with the transcellular route. Our results demonstrated that engulfment of neutrophils and B lymphocytes by fibroblasts resulted in selective passage by a transcellular route.     

Localization of gamma-rays induced chromatid breaks using a three consecutive staining technique.

Three staining techniques (Giemsa, Q-banding and R-banding) are used consecutively to localize the breakage points in chromosomes of human lymphocytes, irradiated during G2-phase with gamma-rays, at doses ranging from 50 to 200 rad. The large majority, about 85% of the breaks, occurs at the interbands, between R- and Q-bands. The discrepancy of this result, with regard to previously reported ones, is attributed to the strong bias of analysis when only one staining technique is used.     

A method for the identification of human peripheral blood T lymphocytes by sequential immunogold and esterase double staining

A double staining method involving the sequential use of monoclonal OKT hybridoma antibodies applied in the colloidal immunogold method and followed by a simultaneously capturing azo dye method for the detection of acid alpha-naphthyl acetate esterase (ANAE) is described. Mononuclear leukocytes isolated from human peripheral blood using a Ficoll-Hypaque density gradient were stained. M-pattern ANAE-positive monocytes (diffuse staining) were excluded from the lymphocyte counts. 80 +/- 5% of all lymphocytes were T-pattern ANAE positive (dot-like staining) and 77 +/- 3% were OKT3 positive. 86 +/- 6% of all ANAE-positive T-pattern lymphocytes were also OKT3 positive, and 89 +/- 6% of all OKT3-positive lymphocytes were also ANAE positive. This indicates that ANAE is a good marker for total human T lymphocytes. 53 +/- 10% of human peripheral blood lymphocytes were OKT4 positive and 87 +/- 8% of all OKT4-positive lymphocytes were also ANAE positive. 30 +/- 6% of all lymphocytes were OKT8 positive, and only 26 +/- 18% of all OKT8-positive lymphocytes were ANAE negative. This indicates that ANAE cannot be used to distinguish T-helper and T-suppressor lymphocytes as identified by monoclonal antibodies.     

Alternative staining methods for Lowicryl sections.

A number of stains and stain combinations have been identified that, when used with the hydrophilic resin Lowicryl K11M, produce marked improvements over aqueous uranyl and lead salts (UA-Pb) in terms of low granularity, specificity, and range of components contrasted. Three test specimens, tobacco mosaic virus (TMV), starfish sperm, and cultured mouse fibroblasts, were used to evaluate stain characteristics. UA-Pb showed a preference for nuclei acids, which were stained specifically by osmium ammine-B at pH 1.5. A number of stain combinations in which UA was followed or preceded by salts containing barium, manganese, tungsten, molybdenum, and vanadium provided excellent staining of protein-containing components, each stain combination being unique in terms of the degree to which specific components were discriminated. These stains were particularly effective for visualizing internal components of the nucleus where a number of fibrillar and particulate structures not seen with UA-Pb were well contrasted.     

Patterns of silver staining in cells of six-day blastocyst and kidney fibroblast of the domestic rabbit

The distribution pattern of silver-NORs was studied in cells of six-day blastocysts and kidney fibroblasts of the rabbit using the Ag-AS technique. At metaphase and interphase there was a binomial distribution of the number of stained sites in both populations but blastocysts had a greater percentage of cells with larger numbers of stained sites. Up to 7 of the 8 chromosomes known to bear NORs were stained in cells from blastocysts while a maximum of 6 were stained in fibroblasts. A significant difference was found between the mean numbers of chromosomal NORs per cell in metaphases from blastocysts and fibroblasts, where they were 4.2 and 3.3 respectively. Similarly, the mean number of NORs in interphase was significantly greater in cells from blastocysts. The distribution of staining on chromosome pair 13 was related to cell type. Significantly more cells in blastocysts than fibroblasts showed staining in this chromosome pair.     

AMC-anti-FITC conjugates: novel reagents for amplified immunochemical techniques. Immunofluorescent staining of human fibroblasts

Summary Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acidN-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining procedures. These methods combine the advantages of the fluorescence properties of AMC and the ready availability of FITC-labelled antisera to provide an amplified fluorescence signal as well as overcoming the photobleaching problems in FITC staining. The method is easy to perform and is expected to make an important contribution to the improvement of the quality of staining achieved with immunofluorescence. Details of the procedure used to stain human fibroblasts with antifibronectin antibodies are reported in order to illustrate the method.     

Analysis of the length of S-phase required to show sister chromatid differential staining

In vitro studies of BrdU-dependent sister chromatid differential staining typically employ two cycles of BrdU incorporation. Experiments are described which determined the actual fraction of both S-phases that the rat embryonic fibroblasts (Rat-1) cells had to traverse in order to show distinctive differential staining. Following synchronization of cells by a combination of serum deprivation and hydroxyurea blockage, sister chromatid differential staining, labelling index, mitotic index, and per cent DNA replication are determined. Results indicate that only approximately 50% of the first S-phase is necessary in order to show distinctive differential staining. The importance of this finding to studies of cellular proliferation using BrdU incorporation is discussed.