Multiple fluorescence in situ hybridization

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.     

Fluorescein conjugates as indicators of subcellular pH. A critical evaluation

Fluorescein and tetramethylrhodamine conjugates to protein or dextran were used to determine subcellular pH. The pH dependence of fluorescence of fluorescein isothiocyanate (FITC) conjugates could be described by a single proton dissociation (pK'a approximately 6.8). This allowed pH to be derived accurately from spectra using the simple Henderson-Hasslebach equation. FITC and TRITC conjugates were delivered to mouse macrophage lysosomes by pinocytosis. Lysosomal pH was then determined in several different ways. First, by direct matching of the subcellular fluorescence spectrum with calibration spectra obtained in free solution. Secondly, monensin was used to equilibrate internal and extracellular pH. Subcellular pH could then be determined by the relative increase in fluorescence of the FITC conjugate without loss of probe from the lysosomes. This allowed the calibration of pH dependence with the probe in situ. Finally, macrophages were permitted to pinocytose FITC and TRITC dextran conjugates simultaneously. pH could be determined from the ratio of emissions from the two dyes within the lysosomes. Each of these different methods gave a similar value for lysosomal pH (4.8 +/- 0.1).     

An improved method for chromosome banding after fluorescence in situ hybridization: Use of a tetramethylrodhamine-conjugated BrdU antibody

A method for high quality chromosome banding after in situ hybridization with biotinylated probes has been developed. Fluoresceine-conjugated avidin is used for probe detection, while chromosome banding is performed with a tetramethylrodhamine-conjugated anti-BrdU antibody. In this way probe localization and chromosome identification can be performed simultaneously simply by changing the incidental light wavelength.Abbreviations BAT BrdU antibody technique - DABCO 1,4 diazobicyclo-(2.2.2)octane - FITC fluorescein isothiocyanate - FPG fluorochrome plus giemsa - PHA phytohemagglutinin - RBA R-banding BrdU acridine - TRITC tetramethylrhodamine isothiocyanate     

Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences

A method is described for visualizing three nucleic acid sequences simultaneously by in situ hybridization using a new blue immunofluorescent label, amino methyl coumarin acetic acid (AMCA), in combination with green and red fluorescing FITC and TRITC. Three chromosome-specific repetitive probes labeled with either amino acetyl fluorene (AAF), mercury, or biotin were hybridized simultaneously to metaphase chromosomes prepared from human blood lymphocytes or to interphase tumor nuclei. Conditions for the combined use of three immunocytochemical affinity systems as well as the optimal spectral separation of the three fluorescing labels have been determined. Three-color in situ hybridization was applied to the study of numerical chromosome abnormalities as occur in human solid tumors. Further applications of this method in prenatal diagnosis for the detection of aneuploidy of the most frequently involved autosomes, as well as for the quantification of gene copy number and mRNA expression, are discussed.     

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.     

Cleavage of pig embryos after labeling with fluorescent dyes

In studies on embryonic development, treated and control ova could be co-mixed before transfer to recipients if nontoxic labels for ova were available. These experiments were conducted to determine whether pig ova would continue to cleave after being stained with the fluorochromes tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC). In the first experiment, pig ova stained with TRITC and unstained control ova were transferred into opposite oviducts of recipient gilts. In the second experiment, ova stained with TRITC and ova stained with FITC were transferred into opposite oviducts of recipient gilts. Embryos were recovered 96 h after transfer (Day 6; Day 0 = onset of estrus), the presence of fluorescence was determined, and the number of nuclei per embryo was assessed. Stained ova retained sufficient fluorochrome to permit detection until the zonae pellucidae were shed. Development of embryos stained with TRITC was equal to that of unstained control embryos. However, development of embryos stained with FITC appeared slightly retarded in comparison to that of TRITC-stained embryos. These findings demonstrate the efficacy of the fluorescent staining technique for pig ova during the first six days of pregnancy.     

Stabilization of Fluorochromes After Immunofluorescent Staining of Tissue and Embedding in Epon

Tissue blocks or sections immunofluo-nt stnined before embedding, ig., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluo-in isotbiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade I- during illumination. This is an advantage of the preembedding, immunofluo-nt staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.     

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.Abbreviations PEP phosphoenol pyruvate - PAG polyacrylamide gel - NADP MDH NADP malate dehydrogenase - FITC fluorescein isothiocyanate - SAB serum albumine bovine - DTT dithiothreitol - MDH malate dehydrogenase - ME malic enzyme - PBS phosphate buffer saline - PAP peroxidase anti-peroxidase     

Stabilization of fluorochromes after immunofluorescent staining of tissue and embedding in Epon

Tissue blocks or sections immunofluorescent stained before embedding, i.g., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade less during illumination. This is an advantage of the pre-embedding, immunofluorescent staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.     

Conjugation of lectins with fluorochromes: An approach to histochemical double labeling of carbohydrate components

Summary Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyanate (TRITC) are reported. 20 g of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were separated on DEAE-Sephadex-A 50. Using two different lectins which were conjugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.     

Two- and three-color immunofluorescence using aminocoumarin, fluorescein, and phycoerythrin-labelled antibodies and single laser flow cytometry.

Antibodies coupled to 7-aminocoumarin (AMCA) emit a bright blue fluorescence under ultraviolet (UV) excitation and are therefore ideal for three-color immunofluorescence (IF) with fluorescein (FITC) and phycoerythrin (PE) labeled reagents; however, due to the different absorption spectra, the use of these fluorophores for multicolor flow-cytometric analysis requires a double light excitation source (e.g., two-laser system). We report a strategy which uses a single argon-ion laser to simultaneously excite AMCA, FITC, and PE, thus allowing the flow cytometric analysis of three immunological parameters. When the UV-visible argon-ion laser is fitted with an appropriate set of mirrors, the 35.1-363.8 nm (UV) and 488 nm wavelengths (accounting for 80 mW and 520 mW, respectively) are simultaneously generated; these lines can then be exactly focused on the same observation point by an achromatic cylindrical lens. A number of comparative analysis were performed with this instrumental set up to verify the sensitivity of AMCA IF and its possible application for multicolor immunophenotypic evaluation of blood cell subsets. When AMCA- and FITC-labeled antimouse Ig antibodies were assessed for their ability to detect limiting amounts of mouse monoclonal antibody bound to cells, the former was less sensitive than the latter. A number of factors, including differences in excitation energy (80 mW for AMCA and 520 mw for FITC) and extinction coefficients (1.9 x 10(4) for AMCA and 6 x 10(4) for FITC) could explain this result.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical localization of neuronal antigens: tyrosine hydroxylase, substance P, [Met5]-enkephalin.

Neuronal antigens can be demonstrated histologically by numerous direct and indirect immunocytochemical techniques in which a specific antibody is identified by a marker compound such as fluorescein isothiocyanate, ferritin, or horseradish peroxidase. One of the more sensitive methods for the light and electron microscopic localizations of antigens in sections of tissue is the peroxidase-antiperoxidase (PAP) technique. The experimental procedures and the results obtained using this technique for the localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase, are described. The cellular and ultrastructural localization of the enzyme is demonstrated in perikarya, processes, and terminals of catecholaminergic neurons in rat brain. The immunocytochemical localization of tyrosine hydroxylase is compared to the localization of two peptides, substance P and [Met5]-enkephalin, in the A2 region of the medulla. These studies suggest that a synaptic interaction exists between the catecholaminergic neurons and neurons showing positive immunoreactivity for the peptides. The limitations of the PAP immunocytochemical technique are also discussed in relation to the immunocytochemical localization of tyrosine hydroxylase and other antigens.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.     

Electrophoretic map of boar sperm plasma membrane polypeptides and localization and fractionation of specific polypeptide subclasses

A high resolution, two-dimensional (2-D) electrophoretic map of the plasma membrane (PM) polypeptides from the ejaculated boar spermatozoon is described. 2-D silver-stained polyacrylamide gel electrophoresis (PAGE) gels revealed over 250 polypeptides; Coomassie blue staining revealed more than 100. Fifty Coomassie-staining polypeptides were catalogued and biochemically characterized, with twenty of these designated major sperm PM polypeptides. Cavitation pressures ranging from 50 PSI to 1000 PSI were used to enrich 2-D maps either in head PM (50 PSI) or in flagellar PM (1000 PSI) and provided tentative localization of certain PM polypeptides. Immunoabsorption chromatography showed that most major polypeptides seen in the 2-D map were antigenic. Major polypeptide bands from single dimensional (1-D) gels were excised, antibodies against them were produced in rabbits, and the polypeptides were localized via indirect fluorescein isothiocyanate (FITC) technique. Cross-reacting antigenic determinants in the PAGE PM polypeptides were determined by transblotting and staining the transblots by an indirect peroxidase technique. Cross-reactivity was extensive with several polypeptide groups, but specific enough with others to allow tentative localization. Lectin affinity chromatography using Con A, WGA, RCA-1, PNA, and DBA indicated the lectin specificity of PM polypeptides. These data together with periodic acid-Schiff (PAS) and carbohydrate-specific silver staining permitted identification of glycoproteins in the 2-D maps. FITC coupled to specific lectins showed the regional distribution of these lectins on the sperm surface. The 2-D polypeptide map and the catalogue of properties of major Coomassie-stained PM polypeptides provides a reference for future studies in the boar and other species.     

Localization of vitellogenin and serum albumin in hepatic parenchymal cells of normal and estradiol-treated immature chickens

The localization of albumin and vitellogenin was determined in liver sections from control and estradiol-treated chickens by two different immunocytochemical techniques: (1) The sandwich technique with rabbit anti-lipovitellin or rabbit anti-albumin IgG and fluorescent goat anti-rabbit IgG and (2) the mixed aggregation immunocytochemical technique with anti-lipovitellin IgG and fluorescent lipovitellin.The results show that the antibody against albumin bound only to all liver parenchymal cells. Furthermore, the fluorescence intensity was equally strong in the portal, intermediate and central zones of the lobules.The fluorescent stain for vittelogenin was not above background in livers of control chicks but was far above background in estradiol-treated chicks. As with albumin the fluorescent stain was distributed equally among the parenchymal cells.The results were quantitatively the same 2 and 4 days after estradiol treatment. The relative rates of synthesis and the concentrations of albumin and vitellogenin correlate well with values obtained for tissue sections by immunocytochemical techniques.     

Heterokaryon Identification Through Simultaneous Fluorescence of Tetramethylrhodamine Isothiocyanate and Fluorescein Isothiocyanate Labelled Protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein iso-thiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emisson spectrum, but not both, was possible for any single filter set.     

Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy.

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible.     

Accessibility of low‐molecular‐mass molecules to the median eminence and arcuate hypothalamic nucleus of adult mouse

Blood‐derived molecules are able to access to the median eminence (ME) and arcuate hypothalamic nucleus (Arc) due to the lack of the blood–brain barrier. In the present study, we examined the accessibility of low‐molecular‐mass (LMM) molecules into parenchyma in the ME and Arc of adult mice by administration of Dextran 3000 (Dex3k), Dex10k, Evans blue (EB) and fluorescein isothiocyanate (FITC). In the external zone of the ME, the fluorescence of Dex3k, EB and FITC tracers generated an intensity gradient from fenestrated capillary, but that of Dex10k was detected only between the inner and outer basement membrane of pericapillary space. The fluorescence of FITC in the external zone of the ME was closely associated with axonal terminals and surrounded by cellular processes of tanycytes‐like cells and astrocytes. In the ependymal/internal zone of the ME and Arc, the fluorescence of all LMM tracers was seen at tanycytes‐like cells and neurons. The fluorescence of EB and FITC in these regions was not detected when brains were fixed during or before the administration of tracers. The inhomogeneity of accessibility for fluorescent tracers depended on routes for tracer administration. Thus, the present study indicates that the accessibility of LMM blood‐derived molecules to parenchyma depends on fenestration of the capillary in the external zone of the ME and active transport of ependymal cells in the ependymal/internal zone of the ME and Arc. Copyright © 2013 John Wiley & Sons, Ltd.     

Inorganic phosphors as new luminescent labels for immunocytochemistry and time-resolved microscopy

A new strongly luminescent marker consisting of inorganic crystals is described for time-resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time-resolved microscopy. The luminescence of these phosphors is strong and non-fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half life-time of approximately 700 musec, upon excitation with near ultraviolet light (360 nm). Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time-resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.