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PubMed2006

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

BACKGROUND: Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. RESULTS: The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. CONCLUSION: RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Animals↗

PubMed1988

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry.

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.

Animals↗

PubMed1980

Quantitative bone histology: a new method.

A method is described for quantitative histology of undecalcified bone using an embedding medium suitable for histochemistry. Six normal subjects were given tetracycline markers 24 and 3 d prior to the biopsy which was taken from the posterior iliac spine using an 8 gauge Jamshidi needle. The tissue was embedded in a mixture of equal parts methylmethacrylate and hydroxyethylmethacrylate. Three consecutive sections were cut and (1) stained histochemically for acid phosphatase activity to identify osteoclast cytoplasm, (2) stained with pyronin to identify osteoblast cytoplasm, (3) mounted unstained for tetracycline fluorescence. Tracings of the 3 sections were superimposed using a camera lucida attachment to the microscope, and the tracings quantitated with an electronic digitizer. The major parameters were bone area (17.1 +/- 4.4%) (mean +/- SD), osteoblast or forming surface (5.9 +/- 2.7%) and osteoclast or resorbing surface (1.0 +/- 0.3%). Bone apposition rate was 1.0 +/- 0.2 micrometers/day and resorption velocity (calculated by assuming bone formation and resorption to be equal) 8.4 +/- 4.1 micrometers/day. Mineralization was assessed by osteoid area (3.8 +/- 2.5%), osteoid surface (13.9 +/- 6.6%), tetracycline uptake at the mineralizing front (62.3 +/- 13.5%), and mineralization lag time (18.1 +/- 2.5 days).

Acid Phosphatase↗

PubMed1982

A simple method of staining juxtaglomerular granules in the rat kidney for high resolution light microscopy.

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 micrometer) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicrotome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 X or a 100 X objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.

Animals↗

PubMed2003

[Study on the kinetic fluorimetric determination of tannins in tea].

A simple and highly sensitive kinetic fluorimetric method is proposed for the determination of trace tannins, based on the activation of tannins on the oxidation of pyronine Y by hydrogen peroxide catalyzed by Cu(II) ion. The effects of some experimental conditions were investigated and discussed in detail. The fixed reaction time procedure was used to determine the fluorescence intensity of the system. The calibration curve of tannin was linear in the range of 0.06-0.96 mg.L-1, and the detection limit for tannin was 0.032 mg.L-1. The relative standard deviation for the measurement of 0.32 mg.L-1 tannin (n = 11) was 2.3%. The proposed method has been successfully applied to the determination of tannins in tea. The results obtained were compared with those provided by the Folin-Ciocalteu method.

Calibration↗

PubMed2006

A novel subpopulation lacking Oct4 expression in the testicular side population.

We characterized murine spermatogonial stem cells (SSCs) using a multi-parameter selection strategy, combining Oct4 expression determined by monitoring green fluorescent protein (GFP) expression, and the testicular side population (SP) showing weak fluorescence on Hoechst 33342 dye staining, as markers of stem cell purification. Testicular cells were collected from Oct4/GFP transgenic mice and analyzed using a fluorescence-activated cell sorter (FACS). SP was detected in testicular cell suspensions at an average rate of 0.10%. Multicolor analysis indicated that 96% of SP cells were negative for Oct4. The cells did not express SSC marker genes, but expressed Bcrp1. While the main population was 93% positive for pyronin Y staining, this was limited to 51% in SP. We found a novel subpopulation with reduced RNA content lacking Oct4 expression in testicular SP. These results suggest that the cells isolated by FACS represent a novel population of SSCs in the G0 quiescent state.

Animals↗

PubMed1989

The antitumor intercalating drug ditercalinium binds preferentially to RNA in Friend erythroleukemia cells.

Ditercalinium (DIT; NSC 335153), a 7H-pyridocarbazole dimer, was reported to be capable of binding with high affinity to DNA by bisintercalation. Both the cytostatic and cytotoxic effects of this drug have been attributed to its binding to DNA. DIT inhibits the growth and is cytotoxic to Friend erythroleukemia (FL) cells. When FL cells were treated with 0.5-2.5 microM DIT and then stained with acridine orange (AO), which differentially stains DNA and RNA, the green, orthochromatic fluorescence representing AO binding to DNA was unchanged, while the metachromatic red luminescence characteristic of AO binding to RNA was reduced by as much as 40% in 4 hr; the effect was DIT-concentration dependent. The reduction in RNA stainability by DIT in the absence of any significant decrease in RNA content, was also observed with another RNA-specific fluorochrome, pyronin Y (PY). These results indicate that in live cells DIT preferentially binds to RNA rather than DNA, preventing stainability of the former by the monointercalating dyes AO and PY. When FL cells were exposed to 10 microM DIT after being first permeabilized by ethanol, the subsequent stainability of DNA in these cells was reduced by up to 67% and RNA by up to 44%, indicating that under these conditions DIT binds to both DNA and RNA. This observation was confirmed by competition experiments between AO and DIT bound to DNA or RNA in permeabilized cells mixed with equivalent numbers of RNA-containing (DNase-treated) or DNA-containing (RNase-treated) cells, respectively. The mechanisms that protect DNA against binding by DIT in live cells are unknown but are lost in fixed cells and may be related to maintenance of cellular and/or nuclear membrane integrity. If the propensity for other intercalating drugs to bind to RNA in live cells is correlated with their antitumor activity as is DIT, the rationale for designing new drugs based solely on their affinity for DNA should be reevaluated.

Acridine Orange↗

PubMedDate not supplied

[Circular dichroism of DNA--dye complexes. II. Anisotropy of the long-wave circular dichroism effect and structure of the complex].

Anisotropy of torsional strength of the splitted electronic transition in the case of chromophore-chromophore interaction of dye molecules situated on the helical matrix was considered theoretically and as analytical expression for the value Rperpendicular/Rparallel was obtained. These theoretical results were compared with the experimental data obtained with DNA-proflavine, DNA-pyronine and DNA-acridine orange complexes oriented in multicappilar flow-cell. Studies of the optical effects (optical density and CD changes) due to orientation of these complexes showed that the acridine chromophores are not perpendicular with respect to the DNA axis (alpha D = 19--22 degrees). The DNA base pairs in complexes as assumed also are not perpendicular to the DNA axis, the inclination angle of their transition moments (for the band near 260 nm) being bigger than that of dye chromophores (24 degrees). These results indicate that under experimental conditions used by us no intercalation can be observed.

Acridines↗

PubMed1981

A rapid procedure for the staining of chromosomes in fixed animal materials.

This paper presents a very simple and reliable procedure for the staining of animal chromosomes employing dyes, such as pyronin G, acridine red, rhodamine B, rhodamine 3GO, belonging to aminoxanthene group and brilliant cresyl blue and methylene violet 3RD, belonging to quinone-imine group. The procedure has been tried on sections of the grasshopper and mouse testes fixed in Dutt's modification of Nawaschin mixture. The method is to deparaffinise sections and then to stain with aqueous solution of these dyes for 2--3 minutes, rinsed with water and dehydrated through grades of ethanol, keeping for 15--30 seconds in each grade with several dips. Preparations are then cleared in xylene and mounted. Stained preparations following this procedure revealed excellent colouration of the chromosomes at all the various stages of mitosis and meiosis, particularly in the case of the grasshopper. Mouse chromosomes stained with these dyes following the same method revealed perfect colouration of the fully condensed chromosomes at all stages of mitosis and meiosis but not of the very early stages, except the sex chromosome. Moreover, grasshopper testis sections when treated with cold concentrated phosphoric acid for varying time-periods and then stained with these dyes also revealed excellent colouration of the chromosomes. The implications of these findings have been discussed.

Animals↗

PubMed1980

[Karyotype in GPG bands: description of a elementaly technique for routine (author's transl)].

A new technic for banded chromosomes, with similar results (100%) to those obtained with chromatids modification by trypsin (G bands), is described. The key consists in Pyronin addition (20 mg.%) to distilled water at hypotonic shock, following then a standard karyotype technic assayed with micro-method, reaching banding after Giemsa tinction at 3% in 6.7 phosphate pH. Its interest radicates in chromosome modification in vivo, simplicity, fiability (80% of excellent results) and reproductibility, allowing any cytogenetic laboratory a routine obtention of G bands in only four days.

Azure Stains↗

PubMedDate not supplied

[Experimental and theoretical studies of the formation of DNA complexes with biologically active ligands containing chromophore groups, depending on the ionic strength of the solution].

The binding of two ligands--dyes pyronin G (PG) and methylene blue (MB)--to DNA depending on the Na+ concentration was studied spectrophotometrically in the visible and UV ranges. The theoretical model taking into account the binding of a dye to DNA by means of aggregation and intercalation on two independent lattices was used for computation of the complexation parameters. The results of optimization of spectrophotometric concentration dependences show that the ligand occupies two phosphates in the process of aggregation on a negatively charged matrix. In is shown that the position of isobestic points can serve as an additional criterion for choosing the best values of the cooperativity parameters, absorption spectra of ligands in the aggregated state, and other parameters of the complex formation. The obtained intercalation constants show a linear dependence of 1gK(int) vs 1g C0Na for both PG and MB. These equations were used in a modified version of the computer program which allows optimization of the absorption spectra for DNA-ligand mixtures in a wide range of Na+ concentrations simultaneously, and estimation of the constant for Na+ binding to DNA phosphate (K1 < 10).

Animals↗

PubMed1976

Identification of monocytes in suspensions of mononuclear cells.

A new histochemical technique for morphological studies of mononuclear cells and granulocytes, based on fluorescent staining with methyl green, pyronine Y, and stilbene-isothiocyanato disulfonic acid (MPS stain) is described. The method was applied to mononuclear cells isolated from the blood of normal human subjects by Ficoll-Isopaque density centrifugation. Three cell populations were distinguished, mainly on the basis of differences in morphology and cytochemistry, utilizing the MPS stain. One of the cell types had many of the morphological characteristics of the monocyte. This technique, augmented by lysosomal content and endocytosis capacity studies, revealed contimination of the cell suspension with a larger percentage of monocytes than has usually been reported in the literature.

Cyanates↗

PubMed2000

Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence.

BACKGROUND: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. METHODS: Nucleic acid staining was performed at low pH in the presence of saponin. DNA was stained with 7-aminoactinomycin D (7-AAD) and RNA with pyronin Y(G) (PY); both dyes were used at low concentrations, and 7-AAD was exchanged with nonfluorescent actinomycin D after DNA staining to minimize fluorochrome-fluorochrome interactions. For cell surface antigen staining, allophycocyanin was combined with pH-independent Alexa488 instead of fluorescein-isothiocyanate (FITC) because FITC is pH sensitive. RESULTS: This method identified cell cycle subcompartments in CEM cells comparable to published results on cell lines using other dyes and staining methods. Measurement of DNA-RNA content in CD8 lymphocyte subsets of human peripheral blood mononuclear cells costimulated with CD3/CD28.2 showed that, after 48 h of stimulation, 80% of CD8(+) T cells were in the proliferative state, whereas 86% of CD8(+) non-T cells remained in G(0). CONCLUSIONS: This technique permits the clear identification of cellular subpopulations by phenotype and assessment of their cell cycle status.

Antigens, CD↗

PubMed1982

A comparative study of Burkitt's and non-Burkitt's "undifferentiated" malignant lymphoma: immunologic, cytochemical, ultrastructural, cytologic, histopathologic, clinical and cell culture features.

Nine patients with "undifferentiated" lymphoma (DUL) of Burkitt's (AMB, four cases) and non-Burkitt's (NB, five cases) types were studied. All specimens stained for a single immunoglobulin light chain indicating a monoclonal B-cell lymphoid proliferation. Striking methyl green pyronine and punctate Oil-Red O staining were demonstrated in both groups. Histologic and cytologic examination demonstrated a lymphoid proliferation with a high mitotic count, a "starry-sky" pattern, prominent cytoplasmic basophilia and vacuolation in both subtypes. The AMB cases showed a predominance of regular nuclei with 3-5 basophilic nucleoli, while the NB cases showed slightly greater nuclear variability with a greater proportion of nuclei containing 1-2 eosinophilic nucleoli. The AMB patients were all children (median, 12 age years) and those with NB were adults (median, age 54 years). The site of presentation in both groups was predominantly extranodal and abdominal. This report emphasizes the considerable immunologic, morphologic, cytochemical and clinical overlap present among the subtypes of "undifferentiated" lymphoma.

Adolescent↗

PubMed1983

True histiocytic lymphoma. A report of four cases.

Clinical, morphologic, cytochemical, immunologic, and ultrastructural features of four cases of true histiocytic lymphoma are described. The neoplastic cells were large, ranging from 20 to 45 mu in diameter with round, folded, or convoluted nuclei, and abundant eosinophilic cytoplasm. They exhibited diffuse nonspecific esterase activity. Diffuse acid phosphatase activity was present in two cases so tested. Muramidase activity was present in half of the cases. Finely granular PAS-positive material was seen in the cytoplasm. Methyl green-pyronin positivity was variable. An occasional neoplastic cell showed erythropagocytosis in one case. Malignant cells either contained no cytoplasmic immunoglobulins (three cases) or had immunoglobulins of multiple classes (one case). Surface markers were studied in two cases; they were absent in one case, and were of multiple classes in another case. Ultrastructurally the neoplastic cells had lysosomal granules in three cases so examined, and phagolysosomes, phagocytized material and residual bodies in one of three cases so studied. Patients ranged in age from 28 to 60 years. Two patients had extralymphatic tumors. Survival of more than 5 years was seen in one patient.

Adult↗

PubMed1983

Lymphomas or leukemia presenting as ovarian tumors. An analysis of 42 cases.

Forty cases of ovarian lymphoma and two of extramedullary leukemia were examined with emphasis on histologic types correlated with age, modes of presentation, operative findings, including frequency of bilaterality and omental spread, clinical course following therapy, and problems in differential diagnosis. Although most cases were referred with diagnoses other than lymphoma (granulosa cell tumor or dysgerminoma, occasionally anaplastic tumor, Krukenberg tumor, or metastatic breast carcinoma), utilization of sections cut at 4 mu and stained with hematoxylin and eosin, or sections stained by the methyl green pyronine (MGP), naphthol-ASD esterase (NASD) or periodic acid-Schiff (PAS) methods helped bring out the lymphoid or hematopoietic nature of the cells. Sixteen patients were under 20 years of age. They had small noncleaved cell lymphoma (undifferentiated Burkitt's and non-Burkitt's, 10 cases), diffuse immunoblastic large cell lymphoma (4 cases), or acute granulocytic leukemia (2 cases). Twenty-six patients were 29 to 74 years of age and had diffuse large cell lymphoma (10 cases), diffuse immunoblastic large cell lymphoma (9 cases), follicular (nodular) lymphoma (6 cases) or small noncleaved cell lymphoma (1 case). Pain with an abdominal or pelvic mass was the most common presentation. Nine tumors were discovered during investigation of other gynecologic complaints. At laparotomy, the tumors in 55% of cases involved both ovaries, and in 64% also involved extragonadal sites (usually omentum, fallopian tubes, or lymph nodes). Seventeen patients had tumor affecting one ovary, seven of these without any evidence of extragonadal spread. Forty-two percent (15) of 37 patients with follow-up were alive after 2 years. Only nine patients survived more than 5 years; two subsequently died of lymphoma. Favorable prognostic features included: (1) FIGO stage IA; (2) unilateral ovarian involvement; (3) focal involvement of one ovary; and (4) follicular (nodular) lymphoma.

Adolescent↗

PubMed1984

ARH-77, an established human IgG-producing myeloma cell line. I. Morphology, B-cell phenotypic marker profile, and expression of Epstein-Barr virus.

Monolayer cultures of ARH-77 cells, a human myeloma cell line propagated in vitro, display a variety of morphologic entities ranging from small lymphocytes to classic plasma cells. The cells show intense pyronin and periodic acid-Schiff affinity but are negative for colloidal iron, sudan black, and naphtol AS-D chloroacetate esterase. The cells exhibit phenotypic markers pertaining to each stage of the B-cell lineage. They fail to display sheep erythrocyte and bovine erythrocyte-IgG antibody complex rosettes, common acute lymphocytic leukemia (ALL) antigens and T-cell antigens, but most cells display surface complement receptors, Ia-like antigens, and surface and intracytoplasmic Ig. Monoclonal antibodies were negative for T-antigens, myelomonocytic cell antigens, leukemia-associated antigens, and BA-1 and OKT-10 antigens. However, 100% of the cells were positive with OKT-9 and B3/25 antibodies that are specific for transferrin receptors. About 50% to 80% of the cells were positive for surface membrane immunoglobulin (kappa IgG) and about 10% to 50% for cytoplasmic immunoglobulin (kappa IgG). Virtually all cells were positive when tested for nuclear Epstein-Barr virus antigens.

Adult↗

PubMed2001

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter.

BACKGROUND: The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. METHODS: Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. RESULTS: The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. CONCLUSIONS: One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents.

Cell Cycle↗