Use of immunogold electron microscopy and monoclonal antibodies in the identification of nuclear substructures

A cytochemical technique for the ultrastructural localization of unique nuclear antigens is reported. Using a post-embedding indirect immunogold labeling procedure, nuclear antigens in electron-dense regions of the nucleus are localized with a minimum of nonspecific staining. Using this technique and indirect immunofluorescence, a panel of antinuclear monoclonal antibodies is shown to recognize preferentially cell cycle-dependent nuclear substructures. The antigenic domains recognized include specific regions in condensed chromatin, interchromatin granules, euchromatin, and chromosomes. The specificity of antigen recognition is demonstrated with qualitative and quantitative immunogold electron microscopy and immunoblot analysis. These results reveal the existence of previously undefined supramolecular organization within the nucleus and demonstrate the utility of the immunogold procedure when monoclonal antibodies are used.     

Immunobiochemical characterization with monoclonal antibodies of Epstein-Barr virus-associated early antigens in chemically induced cells.

Five monoclonal antibodies which are reactive to early antigens of Epstein-Barr virus have been produced by using somatic cell hybridization techniques. The specificity of the monoclonal antibodies to early antigens was demonstrated by indirect immunofluorescence, which showed that the antigens were localized to the nucleus of early antigen-induced Raji cells. Additional indirect immunofluorescence studies showed that like patient antisera to diffuse-staining early antigen, the monoclonal antibodies gave positive staining reactions after methanol fixation. One of the antibodies, 1150-4, was positive by the anti-complement immunofluorescence technique but differed with Epstein-Barr virus-associated nuclear antigen-positive patient sera in that it only stained induced cells. Different fixation methods were found to alter dramatically the appearance of the nuclear staining reactions produced by the monoclonal antibodies. Immunoprecipitation and immunoblot experiments revealed that monoclonal antibodies 1108-1 and 1129-1 recognized two polypeptides of 55,000 and 50,000 daltons (p55;50), 1173-6 and 1180-2 recognized just p50, and 1150-4 identified a 65,000-dalton nuclear protein. Immunobiochemical characterization of these viral antigens showed that p55 is a phosphoprotein, and p55;50 has strong DNA-binding activity preferentially to single-stranded DNA. Elucidation of the role of these nuclear proteins in Epstein-Barr virus infection and the events associated with Epstein-Barr virus-directed lymphocyte transformation may provide significant information on the pathogenicity of this important human virus.     

Assessment of cell cycle-associated antigen expression using multiparameter flow cytometry and antibody-acridine orange sequential staining

A novel approach which enables direct assessment of the differential expression of cellular antigens in noncycling (G0) and cycling cell subpopulations is presented. The method involves flow cytometric analysis and sorting of cells stained by use of indirect immunofluorescence, followed by restaining using acid acridine orange, to relate the immunofluorescence of sorted lymphoid subpopulation(s) to cell proliferation status (i.e., G0 vs. G1 vs. S vs. G2 and M). In the present study, this technique successfully identifies the proliferation-associated modulation of a heterochromatin-associated antigen in pokeweed mitogen-stimulated human lymphoid cultures. The potential utility of this method for documenting early antigenic changes associated with the G0-G1 transition is discussed.     

盐酸酸化可改善核抗原免疫荧光染色

免疫荧光染色(immunofluorescent staining, IF)技术广泛用于细胞或组织内抗原定性、定量或定位检测。然而,依常规染色步骤操作,在有些胞核抗原的检测中很难得到令人满意的结果。有研究者采用盐酸酸化预处理用于细胞增殖标记物5-溴-2-脱氧尿嘧啶核苷(BrdU)的荧光染色并获得良好效果。但此方法是否也适用于其他类型的胞核抗原,尚不清楚。为系统全面分析盐酸酸化在胞核抗原免疫荧光染色中的作用,本文以成年C57BL小鼠主要嗅觉表皮(MOE)为材料,分别对Ki-67、5-甲基胞嘧啶(5mC)和5-羟甲基胞嘧啶(5hmC)三种不同类型的胞核抗原进行盐酸酸化处理免疫荧光染色。结果显示,当血清封闭和抗体浓度等条件一致时,室温下盐酸酸化2 h,Ki-67的染色效果最佳,而阴性对照与未酸化组均未出现阳性信号;同样经过盐酸处理2 h,5mC和5hmC染色也呈现较强的阳性信号。该研究表明,在一些胞核抗原免疫荧光染色中,使用盐酸酸化处理可显著提高染色效果。     

Flow cytometric prescreening of cervical smears.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.     

Immunohistochemical localization of cyclic GMP in aggregating Polysphondylium violaceum.

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.     

Immunohistochemical Localization of Cyclic GMP in Aggregating Polysphondylium violaceum

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.     

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.     

Immunolocalization of membrane-associated CTP:phosphocholine cytidylyltransferase in phosphatidylcholine-deficient Chinese hamster ovary cells.

The location of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary (CHO) cells made deficient in phosphatidylcholine was determined by immunofluorescence techniques. A rabbit polyclonal antibody was raised against a synthetic peptide corresponding to the amino-terminal 17 amino acid residues of rat liver cytidylyltransferase. The antibody recognized both native and denatured cytidylyltransferase from both rat liver and CHO cells. CHO cells were treated with phospholipase C to alter the lipid composition of the plasma membrane and to elicit translocation of cytidylyltransferase from the less active soluble pool to an activated membrane fraction. Visualization of cytidylyltransferase by indirect immunofluorescence revealed staining of the nuclear envelope in phospholipase C-treated cells but not in untreated cells. CHO cells were also starved for choline and supplemented with a choline analogue to provide an alternative technique of rendering the cells phosphatidylcholine-deficient. Although this treatment should affect different cellular membranes than those affected by phospholipase C treatment, cytidylyltransferase still translocated to the nuclear envelope, as shown by indirect immunofluorescence. These results indicate that activated, membrane-bound cytidylyltransferase is associated with the nuclear membrane and suggest that the nuclear membrane may be a site of de novo phosphatidylcholine synthesis.     

A spectrin-like protein present on membranes of Amoeba proteus as studied with monoclonal antibodies

Monoclonal antibodies against a spectrin-like membrane-associated protein of xD amoebae. (Amoeba proteus) were used to determine the distribution of the protein and some of its characteristics. A total of 34 monoclonal antibodies recognizing different epitopes of the protein were obtained, of which seven stained cell membranes by indirect immunofluorescence. The spectrin-like protein had two subtypes of 225 and 220 kDa and several monoclonal antibodies cross-reacted with human erythrocyte spectrin when checked by indirect immunofluorescence staining and immunoblotting. Some of the antibodies also cross-reacted with antigens in HeLa cells and chick embryo fibroblasts. Polyclonal and monoclonal antibodies against Drosophila and human erythrocyte spectrins cross-reacted with the spectrin-like protein from amoebae. On the basis of these results, it was concluded that the protein is a spectrin. The protein was found on most cellular membranes of amoebae, including the plasma, nuclear, and phagosomal membranes, as well as symbiosome membranes.     

Indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins in human A431 cells.

Polyclonal antibodies directed against (i) rodent lung beta 2-adrenergic receptor, (ii) a synthetic fragment of an extracellular domain of the receptor, and (iii) human placenta G-protein beta-subunits, were used to localize these antigens in situ in intact and permeabilized human epidermoid carcinoma A431 cells. Antibodies directed against beta 2-adrenergic receptors showed a punctate immunofluorescence staining throughout the cell surface of fixed intact cells. Punctate staining was also observed in clones of Chinese hamster ovary cells transfected with an expression vector harbouring the gene for the hamster beta 2-adrenergic receptor. The immunofluorescence observed with anti-receptor antibodies paralleled the level of receptor expression. In contrast, the beta-subunits common to G-proteins were not stained in fixed intact cells, presumably reflecting their intracellular localization. In detergent-permeabilized fixed cells, strong punctate staining of G beta-subunits was observed throughout the cytoplasm. This is the first indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins. Punctate immunofluorescence staining suggests that both antigens are distributed in clusters.     

Quantitative measurement of SV40 T-antigen production

African green monkey cells (CV-1) were infected with SV40 virus at high multiplicities of infection (MOI), and the production of T-antigen was studied. A new instrumentation, flow microfluorometry, coupled with indirect immunofluorescence permitted quantitative evaluation of this antigen. Optimum conditions were determined for antibody excess. Antigen production was not detected for the first 6 h post-infection. The value of this technology is discussed in relation to quantitative evaluation of expression of cellular antigens.     

Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2.

About 50% of Semliki Forest virus-specific nonstructural protein nsP2 is associated with the nuclear fraction in virus-infected BHK cells. Transport into the nucleus must be specific, since only trace amounts of nsP3 and nsP4 and about 13% of nsP1, all derived from the same polyprotein, were found in the nucleus. Subfractionation of [35S]methionine-labeled Semliki Forest virus-infected cells showed that 80 to 90% of the nuclear nsP2 was associated with the nuclear matrix. Indirect immunofluorescence, with anti-nsP2 antiserum, showed the most intensive staining of structures which by Nomarski optics appeared to be nucleoli. In the presence of 1 to 5 micrograms of dactinomycin per ml the nuclei were stained evenly and no nucleoli could be found. Transport of nsP2 into the nucleus occurred early in infection and was fairly rapid. A cDNA encoding the complete nsP2 was isolated by the polymerase chain reaction technique and ligated into a simian virus 40 expression vector derivative. When BHK cells were transfected with this pSV-NS2 vector by the lipofection procedure, nsP2 was expressed in about 1 to 5% of the cells, as shown by indirect immunofluorescence. In positively transfected cells the immunofluorescence stain was most intensive in the nucleoli. Thus, Semliki Forest virus-specific nsP2 must have information which directs it into the nuclear matrix and, more specifically, into the nucleoli.     

Correlative immunofluorescence and electron microscopy on the same section of epon-embedded material

Semithick (0.25-0.50 micron) sections, cut from cells stained with fluorescein isothiocyanate (FITC)-conjugated antibodies prior to embedding in Epon, show high resolution patterns of immunofluorescence against a background void of autofluorescence. These same sections can then be viewed, after uranyl and lead staining, in the electron microscope. We clearly establish the specificity of this same-section correlative immunofluorescence-electron microscopy approach by showing that the immunofluorescent patterns observed in such sections of cells, stained prior to embedding for the indirect immunofluorescent localization of tubulin, follows the distribution of microtubules within the same sections as determined by electron microscopy. We then use this method to demonstrate for the first time that the 57 kD core protein of wound tumor virus is associated, at the ultrastructural level, with two distinct cellular inclusions in virally infected AC-20 cells. In some instances the fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section eliminates the need to employ more complex procedures for labeling antigens for ultrastructural detection. This technique, therefore, provides a rapid and simple first approach to many problems that require the ultrastructural localization of specific antigens.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

Proacrosin and the differentiation of the spermatozoa

Using the indirect immunofluorescence staining technique, the occurrence and localization of proacrosin, the zymogen form of acrosin, was studied during spermatogenesis in the bull, ram, boar and rabbit. Proacrosin staining was demonstrable for the first time in the early haploid spermatid and increased with the differentiation of the spermatid to spermatozoon. The spermatozoon is covered by a cap-like structure of uniform fluorescence corresponding to the acrosomal compartment of the male gamete. No fluorescence could be found in diploid spermatogenic cells, i.e., in spermatogonia and spermatocytes. An identical developmental pattern of proacrosin was observed with the indirect immunoperoxidase staining technique. However, with this staining technique a distinct distribution of proacrosin staining was observed in the acrosome of epididymal and ejaculated spermatozoa of the bull, ram, boar, rabbit and man. Proacrosin seems to be distributed in the acrosome in granules rather than in the homogeneous form, as was indicated by the results of indirect immunofluorescence staining.     

Quantitative immunofluorescence in single Saccharomyces cerevisiae cells

We have developed a staining procedure that allows the simultaneous determination of intracellular amounts of DNA and an antigen in Saccharomyces cerevisiae with a single laser flow cytometer. The antigen, beta-galactosidase from a cloned lacZ gene, is inducible and is detected with an indirect immunofluorescent stain. Cell preparation procedures, specifically cell fixation and cell wall removal, have significant effects on measured levels of immunofluorescence and have been optimized to prevent cell loss and maximize immunofluorescence. Average immunofluorescent levels of cell populations expressing different levels of beta-galactosidase show excellent correlation with measurements of average beta-galactosidase activity per cell based on cleavage of o-nitrophenyl-beta-D-galactopyranoside. Experiments with yeast populations containing various numbers of copies of the cloned gene indicate that the relationship between immunofluorescence and antigen content also holds at the single-cell level. Correlated measurements of DNA and beta-galactosidase content on a single-cell level permit the investigation of cellular enzyme content as a function of cell cycle position under various conditions. The procedure can be easily modified to detect other antigens by changing the primary antibody used.     

Identification of a nuclear protein component of interchromatin granules using a monoclonal antibody and immunogold electron microscopy

A monoclonal antibody, designated 780-3, has been generated which preferentially recognizes an antigenic component of interchromatin granules in human cells. By indirect immunofluorescence procedures, monoclonal antibody 780-3 produces a cell cycle-specific speckled nuclear staining pattern in adult human fibroblasts which is dramatically altered during metaphase. In contrast, transformed cells appear to express this antigen throughout the cell cycle in increased quantities. Immunogold electron microscopy revealed that the nuclear antigen is intimately associated with interchromatin granules in human cells. Analysis by immunoblot procedures showed that monoclonal antibody 780-3 recognizes two polypeptides of 105 and 41 kD. From these data, a possible nucleolar derivation of interchromatin granules is discussed. These studies demonstrate for the first time that monoclonal antibodies may be used in combination with immunogold electron microscopy to identify the ultrastructural location of nuclear antigens.     

Simultaneous staining of nuclear DNA and blood group cell surface antigens on cells from bladder irrigation fluid

Simultaneous staining of nuclear DNA and blood group cell-surface antigens is proposed as a means of studying the prognosis of superficial bladder cancer. The quality of the results obtained on urothelial cells from bladder irrigation fluid, as shown by fluorescence microscopy, suggests that this staining technique may be suitable for flow cytometry.     

Cell surface antigens reacting with antiretrovirus sera on normal mouse spleen cells: a flow cytometric study

The development of erythroleukemia in Balb/c mice injected with Rauscher leukemia virus has been followed by indirect immunofluorescence technique and flow cytometry, using antiserum against disrupted of virions. The progression of the disease was accompanied by a great increase in the number of large, immunofluorescence positive cells. A subpopulation of normal spleen cells was also highly positive. The expression of the antigens in normal cells was examined in relation to the cell cycle. The majority of the S-G2/M phase cells were found in the antigen positive compartment of larger cells. A two-color analysis of immunofluorescence and DNA content revealed that the distribution of antigen expression in G1 phase was broad, gradually decreasing from a low-intensity mode. The cell with double DNA content distributed evenly around a modus of five-fold higher intensity. In experiments with stimulated bone-marrow cells, superiority of S-phase cells in anti-Rauscher serum binding was found. Cell-surface gp70 antigen is suggested to be involved in this cell-cycle dependent binding of antibodies by normal cells.