Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.     

A new infrared fluorescent-labeling agent and labeled antibody for diagnosing microcancers

PURPOSE: We have developed infrared fluorescent labeling agents and infrared-ray fluorescence endoscopes to establish a novel diagnostic technique. Since the fluorescence intensity of the initial labeled antibody (ICG-sulfo-OSu-labeled antibody) was not sufficient for practical use, we synthesized indocyanine green acylthiazolidinethione (ICG-ATT), which was expected to label various target molecules having amino groups efficiently. MATERIALS AND METHODS: To confirm imaging of infrared fluorescence intensity of ICG-ATT- and ICG-sulfo-OSu-labeled anti-MUC1 antibodies, cotton thread was soaked in various concentrations of the antibody solution in 0.1M PBS, and observed under the epi-illumination infrared fluorescence microscope. Localization and the intensity of infrared fluorescence and DAB coloring was compared in paraffin sections of human gastric mucosa. RESULTS: In the study of cotton threads, both labeled antibodies showed relatively clear infrared fluorescence, and significant difference was not observed between the two antibodies. ICG-ATT-labeled anti-MUC1 antibody produced stronger staining than that by ICG-sulfo-OSu-labeled antibody. Localization pattern of infrared fluorescent staining was in good agreement with that by the conventional method with oxidized DAB staining. CONCLUSION: ICG-ATT is useful as a fluorescent-labeling agent for diagnosis of microcancers by infrared fluorescence endoscopes.     

A single laser method for subtraction of cell autofluorescence in flow cytometry

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.     

Detection of myelination using a novel histological probe.

Current methods for myelin staining in tissue sections include both histological and immunohistochemical techniques. Fluorescence immunohistochemistry, which uses antibodies against myelin components such as myelin basic protein, is often used because of the convenience for multiple labeling. To facilitate studies on myelin, this paper describes a quick and easy method for direct myelin staining in rodent and human tissues using novel near-infrared myelin (NIM) dyes that are comparable to other well-characterized histochemical reagents. The near-infrared fluorescence spectra of these probes allow fluorescent staining of tissue sections in multiple channels using visible light fluorophores commonly used in immunocytochemistry. These dyes have been used successfully to detect normal myelin structure and myelin loss in a mouse model of demyelination disease.     

Chromogen-based immunohistochemical method for elucidation of the coexpression of two antigens using antibodies from the same species

The authors established a chromogen-based, double immunolabeling method using antibodies from the same species without any unwanted cross-reactivity. In addition, time-consuming staining steps were shortened by using polymer-based secondary antibodies. Taking advantage of the nature of the chromogen 3-amino-9-ethylcarbazole (AEC), which is used as a horseradish peroxidase substrate for antibody detection, the AEC-derived signals in the first color development were easily eliminated by alcohol treatment. Therefore, the signals from the first staining did not interfere with those from the subsequent second staining, which used the chromogen 3,3'-diaminobenzidine. The co-localization of antigens within the same cell could be confirmed using this method, because cell images of the individual dye staining steps could be obtained and developed. The images from each step could be expressed in pseudo-colors in a dark field by using a computer. As a result, merged images could be constructed that resembled the images acquired by the fluorescent immunolabeling technique. The resolution of this method enabled analysis of the coexpression of two antigens in the same cell in the same section. The authors have named this staining technique the elucidation of the coexpression of two antigens in a cell using antibodies from the same species (ECSS).     

A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents,Immunofluorescence, and Multiple Antibody Staining

Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.     

Modulation of mouse preimplantation development by epidermal growth factor receptor antibodies,antisense RNA,and deoxyoligonucleotides

Two-cell mouse preimplantation embryos were cultured for 48 h in four different reagents to modulate epidermal growth factor (EGF) receptor function. These were rabbit polyclonal and mouse monoclonal antibodies to EGF receptor, EGF receptor antisense RNA, and EGF receptor antisense deoxyoligonucleotides. Embryos were scored for two endpoints: onset of cavitation as a measure of trophectoderm differentiation and mean embryo cell number as a measure of cell proliferation. The consistent observations were that cavitation was significantly accelerated by antibodies and delayed by antisense RNA and antisense deoxyoligonucleotides. None of these reagents exerted a significant effect on mean embryo cell number, with one exception the polyclonal antibody. Our interpretation of these observations is that the antibody binding facilitated cavitation by mimicking natural ligand-receptor binding and inducing the signal transduction cascade that is typical for the EGF receptor. In the case of antisense RNA or deoxyoligonucleotide, we propose that they delayed onset of cavitation by interfering with EGF receptor production. We hypothesize that during this period of development, EGF receptor is concerned predominantly with the regulation of differentiation more than with cell proliferation. © 1993Wiley-Liss, Inc.     

Identification of hemoglobin types contained in single chicken erythrocytes by fluorescent antibody technique

It has been suggested that the switch in hemoglobin (Hb) types (from embryonic to adult) during chicken embryonic development is associated with the substitution of one erythroid cell line (“primitive”) for another (“definitive”). For the detection of two Hb types inside single erythroid cells, rabbit antibodies specific for embryonic and adult Hbs were prepared. Rabbit antibody specific for embryonic Hb cross-reacted only with embryonic major Hb components, while antibody specific for adult Hb did solely with adult minor Hb component. The antibodies were conjugated with fluorescein isothiocyanate. The conjugated antibodies were used for the fluorescent staining of blood smears of developing chicken embryos at different ages. Direct fluorescent antibody technique demonstrated that the major components of embryonic Hb and the minor component of adult Hb were not present within the same erythrocyte during chicken ontogenesis. It strongly suggested that embryonic-type Hb and adult-type Hb do not coexist within the same cell.     

Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum

We have obtained a monoclonal antibody to 80 kD protein of calf serum; this protein easily and uniformly adsorbs on glass from serum-containing media. Indirect immunofluorescence staining of chick and mouse embryo fibroblasts cultured in the presence of calf serum, fixed with formaldehyde and permeabilized with Triton X-100, revealed black non-fluorescent strips and dots under the ventral cell surface, whereas all other parts of the substratum under and between cells were highly fluorescent. The distribution of non-fluorescent regions coincided with the distributed of focal contacts of cells with the substratum, revealed by interference reflection microscopy, as well as with the distribution of vinculin-containing plaques. The dark regions were also associated with the ends of microfilament bundles revealed by immunofluorescence with an anti-actin antibody. Thus, non-fluorescent regions seen after anti-80 kD staining are parts of the substratum under the focal contacts. Visualization of focal contacts with anti-80 kD provides very contrasting and high resolution pictures. Evidence is presented that 80 kD protein is adsorbed to glass in the areas of focal contacts, but the antibodies used for staining cannot penetrate these contacts.     

New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.     

The capping of lymphocytes and other cells, studied by an improved method for immunofluorescence staining of frozen sections.

Experiments have been carried out on the capping by lectins and antibodies of surface receptors of mouse splenic T and B lymphocytes and other cells, in which the surface distribution of the lectin or antibody, and the intracellular distribution of myosin or actin, were determined on the same cells by a double fluorescence technique. For this purpose, a general method for intracellular staining was developed which is intended to preserve sensitive antigens and fragile ultrastructural elements. The method involves mild formaldehyde fixation of the cells or tissues, infusion with concentrated sucrose, rapid freezing, and the preparation of frozen sections thinner than 1 micrometer thickness. The immunofluorescent or other appropriate fluorescent reagents are then applied to the thawed section. In the present experiments, intracellular actin was detected using a fluorescent staining method based on the interaction of F-actin with heavy meromyosin, while intracellular myosin was detected by an indirect immunofluorescence procedure. Our findings were that the formation of a cap by each of the lectins or antibody reagents was always accompanied by a concentration of myosin and actin directly under the cap. These and other results suggest that capping is an active process in which actin and myosin participate directly in the formation of all caps. This proposal carries important new implications for the molecular mechanism of capping.     

Cellular Concentrations and Uniformity of Cell-Type Accumulation of Two Lea Proteins in Cotton Embryos

The levels and cell-type distribution of late embryogenesis abundant (Lea) proteins D-7 and D-113 have been determined in mature cotton embryos by immunochemical methods. The two proteins were expressed in and purified from Escherichia coli and utilized for antibody production in rabbits. The antiserum to each protein was found to interact with all members of each protein family in cotton extracts by protein gel blotting. Using these antibodies in quantitative "rocket" immunoelectrophoreses, D-7 proteins were found to accumulate to ~8 x 1015 molecules per embryo, which is equivalent to ~109 molecules per "average cell." D-113 proteins accumulate to ~1016 molecules per embryo, which equates to ~1.3 x 109 molecules per average cell. These values calculate to concentrations of about 226 and 283 [mu]M, respectively, in the cell aqueous phase immediately prior to seed desiccation. In immunocytochemical studies using the fluorophor rhodamine linked to the secondary antibody, both proteins appeared to be evenly present in the cytosol of all cell types present in the embryo, including both cotyledon and axis epidermal cells. Thus, their function does not appear related to unique functions of specific cell or tissue types. The very high molar concentrations of the two proteins, coupled with their unusual predicted structure and their cytosol location, would seem to reduce the number of their conceivable functions.     

Antibodies introduced into living cells with liposomes localize specifically and inhibit specific intracellular processes

We have developed a system for efficiently packaging antibodies and other macromolecules into lipsomes and then delivering the encapsulated molecules into living cells through liposome-cell fusion. Fusion is very efficient, and all cells can be demonstrated to contain liposome-delivered antibodies by staining by staining with a fluorescent second antibody. Using lupus antibodies directed against small nuclear ribonucleoprotein components of the cell, we were able to demonstrate strong nuclear localization, while control antibodies showed a general diffuse distribution throughout the cell. Lupus antibodies directed against ribosomes, on the other hand, strongly localized in the nucleolus and the cytoplasm with very little nucleoplasmic localization. Antitubulin antibodies predominantly localized in the cytoplasm. These results show that antibodies can survive liposome packaging and can retain their ability to recognize and bind to their specific antigens in the living cell. It also indicates that the nuclear envelope does not present a barrier to the liposome-introduced antibodies in Drosophila tissue culture cells. To determine if the antibodies were capable of interfering with cellular processes in vivo, we measured the effects of liposome-introduced antiribosome antibodies on translation and antitubulin antibodies on mitosis. In both cases, there was a significant inhibition suggesting that the antibodies can be used to interfere with specific functions at specific times in vivo.     

Monoclonal antibodies for diagnosis of immunodeficiencies

Progress in immunophenotyping is characterized by the availability of monoclonal antibodies and an increased number of clusters of differentiation consisting of reagents with known specificity and defined reactivity patterns. Technical improvements have lead to standardization of immunofluorescence staining procedures and broad application of flow cytometry. These developments have contributed to better diagnosis of immunodeficiencies characterized by the lack of certain lymphocyte subsets or more broadly expressed, functionally important cell-surface molecules. Antibodies valuable for routine immunophenotyping of immunodeficiencies as well as examples of the different antibody groups desirable for immunofluorescence studies are presented. When used in concert with clinical and other laboratory tests, immunophenotyping provides a valuable instrument for differential diagnosis of defects in the immune system. As a consequence, detection of new defects of cell surface antigens and respective cell subpopulations is facilitated and a basis is provided for further study of the genetic and molecular regulatory aspects of immunologic disorders.     

Immunohistochemical detection of the CXCR4 expression in tumor tissue using the fluorescent peptide antagonist Ac-TZ14011-FITC

Pathology is fundamental in grading, staging, and treatment planning of malignancies. One relatively novel biomarker that may become more important in therapy and diagnostics is the chemokine receptor 4 (CXCR4). Ac-TZ14011 peptide derivatives, functionalized with a radiolabel, can be used for molecular imaging of tumors. Direct fluorescent labeling of the small peptide Ac-TZ14011 with the fluorescent dye fluorescein isothiocyanate (FITC), however, provides an alternative for the detection of CXCR4 expression levels in cells and tumor tissue. In this study, Ac-TZ14011-FITC was validated for CXCR4 staining in human breast cancer cell lines MDAMB231 and MDAMB231(CXCR4+) during flow cytometric analysis. Its efficacy was compared to commercially available antibodies. Competition experiments validated the staining specificity. Confocal imaging revealed that CXCR4 staining was predominantly found on the cell membrane and/or in vesicles formed after endocytosis. Next to being able to differentiate "high" and "low" CXCR4-expressing tumor cells, the fluorescent peptide demonstrates potential in fluorescent immunohistochemistry of tumor tissue. Ac-TZ14011-FITC was able to differentiate MDAMB231 from MDAMB231(CXCR4+) tumor cells and tissue, proving its applicability in the detection of differences in CXCR4 expression levels.     

Heterogeneity of macrophage populations in human lymphoid tissue and peripheral blood

Human lymphoid tissue and peripheral blood leukocytes were stained with six monoclonal antibodies directed against monocyte/macrophage populations. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages. The results show that there exists considerable heterogeneity of tissue macrophages based on the expression of surface and/or cytoplasmic antigens; furthermore, the distribution of cells bearing particular antigenic determinants is associated with distinct regions in normal lymphoid tissue. Double staining methods demonstrated that these antibodies bind to different, as well as to identical, macrophage populations. OKM-1 antibody binds predominantly to sinus histiocytes and tingible body macrophages. The Leu M-1 reagent stains interdigitating reticulum cells, while the KiM-4 antibody labels follicular dendritic cells. Leu M-3 antibody identifies cells predominantly in the germinal center, and histiocytes lining the sinuses. Both CM-1 and BRL-M.1 appear to stain tissue macrophages distributed throughout the lymphoid tissue.     

Considerations for establishing the validity of immunocytological studies.

Immunocytology has wide spread applications for localizing tissue antigens, as evidenced by the recent exploitation of this technique in biological studies. Documenting the immunological specificity of the staining reaction is one of the most important technical considerations in validating the accrued data in immunocytological studies. The purpose of this report is to discuss and emphasize the need for conducting physiological studies in addition to the traditional immunological method and specificity controls. The ability of antibodies to bind molecules other than those molecules used as the immunizing material is a well documented fact. Hypothetically, preabsorption of the primary antibody with its specific antigen, could reduce subsequent binding of this antibody to a cross reactive tissue antigen, thus providing false confirmation of staining validity. The results of our experience with a cross reacting system in addition to other previously reported examples are discussed.     

Immunohistochemical detection of subunit A(M) of lactate dehydrogenase in tissues of the developing chick

Summary The distribution of A(M) subunits of lactate dehydrogenase (mainly LDH₅) in developing muscle, heart, liver, lung, kidney and cartilage tissue of chicken embryos was examined by the indirect fluorescent antibody technique. Antibodies against porcine LDH₅, purified by affinity chromatography, were used for this purpose. In special areas of newly formed myofibrils in somitic myoblasts fluorescence was already detected after 4 days of incubation, and located at the same place in muscle tissue of all advanced developmental stages examined. During the myotube stage of muscle development staining was also located in the peripheral thickened cytoplasma of the myotubes. The myocardium did not exhibit any fluorescent staining in the developmental stages examined. Endocardium, epicardium and pericardium, however, were fluorescent in young developmental stages. The liver showed fluorescence in 5- to 8-day embryos mainly in the endothelial cells of the blood sinusoids. In 9- to 12-day embryos the bile ducts became fluorescent. In lungs after 9- to 12-day development the epithelium and the surrounding tissues of bronchi exhibited strong immunofluorescence. The mesonephros exhibited faint granular fluorescence in tubule-forming cells and their membranes after 4–9 days of incubation. Advanced developmental stages only exhibited fluorescent blood cells. This latter staining is at least partly due to non-specific reactions of blood cell membranes with FITC-conjugated anti-rabbit IgG. Cartilage is characterized by non-specific fluorescence, but in embryos older than 8 days strong granular fluorescence of chondrocytes and staining of the perichondrium distinguished sections treated with anti-LDH₅ antibodies from control sections reacted only with FITC-conjugated anti-rabbit IgG. In addition, strong fluorescent staining was detectable in certain areas of the 5-day neural tube and faint staining in the mucosa of the intestine from embryos older than 10 days.     

Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglion cells

The monoclonal antibody technique was used to investigate neuronal heterogeneity and its developmental changes in the chick embryo trunk especially at the thoracic level. We report here four monoclonal antibodies (called SC 1, SC 2, SC 3, and SC 4) that bound to cell surface antigens. These antigens appeared to be proteins or glycoproteins because of their susceptibility to trypsin. In the spinal cord, antibody SC 3 stained all cells, but antibody SC 1 specifically stained motoneurons and ventral epithelial cells. The staining of motoneurons by antibody SC 1 was transient. It appeared at early stages (stage 16-17; Hamburger and Hamilton), but decreased markedly in intensity at older stages (stage 30-31). Antibody SC 2 did not stain cells in the spinal cord. It stained only neurons in the dorsal root and sympathetic ganglia. Antibody SC 4 stained only cells derived from the neural crest at the early stages (stage 16-20). At later stages, it stained a wider population of cells, including sensory neurons, Schwann cells, and cells in the central nervous system. In the dorsal root ganglion, antibodies SC 1 and SC 2 stained only neuronal cells whereas antibodies SC 3 and SC 4 stained both neuronal and glial cells. The dorsal root ganglionic antigens recognized by these antibodies were not expressed concurrently but appeared in a developmental sequence. Staining with antibodies SC 3 and SC 4 appeared first, then SC 1, and finally SC 2. Among these four antigens, the antigens common to both neuronal and glial cells appeared earlier than the neuron specific antigens. Thus, our monoclonal antibodies revealed heterogeneities in cell surface neuronal molecules and their transient and sequential appearance during embryonic development.