Endogenous polyamines are intimately associated with highly condensed chromatin in vivo. A fluorescence cytochemical and immunocytochemical study of spermine and spermidine during the cell cycle and in reactivated nuclei

Polyamines are low molecular weight aliphatic polycations essential for cell proliferation and differentiation. By immunocytochemistry, as well as by two independent fluorescence cytochemical methods, we show that polyamines are associated with highly condensed chromatin in nucleated erythrocytes and in metaphase and anaphase chromosomes. In other cells, polyamines mainly occur in cytoplasm. The association between polyamines and DNA in condensed chromatin is so close that DNase treatment is necessary for making polyamines available for reaction with antibodies. Studies of chick/HeLa cell heterokaryons reveal that polyamines disappear from the chick erythrocyte nuclei concomitantly with DNA decondensation and initiation of RNA synthesis. Our data strongly suggest that polyamines are important for chromatin condensation in vivo.     

Localization and possible function of polyamines in protein and peptide secreting cells

Three different cytochemical methods, the formaldehyde-fluorescamine and the ortho-phtalaldehyde fluorescence cytochemical methods as well as immunocytochemistry have been developed for localising the polyamines spermidine and spermine in tissue. All three methods produce identical results and show polyamines to occur inter alia in high concentrations in certain protein- and peptide-secreting cells. Many of these cells also show the capacity to metabolise monoamines and belong to the amine content or amine precursor uptake and decarboxylation (APUD) series. In cell types where fluorescence microscopical resolution allows, most polyamines appear to be localised to secretory granules. Moreover, studies on isolated pancreatic islets reveal active and glucose-dependent polyamine biosynthesis to occur in insulin cells. Possible function of polyamines in secretory granules are discussed in the light of the above findings.     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Summary Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.In honour of Professor P. van Duijn     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.     

Polyamine cytochemistry: Comparisons between cytochemical, autoradiographic, immunocytochemical and chemical results in the prostate

Summary Results obtained with two newly developed fluorescence cytochemical methods for detecting the polyamines spermidine and spermine have been compared to autoradiographic localization of biosynthetically labelled polyamines, to immunocytochemical results obtained with antibodies directed against spermidine and spermine, and to chemical polyamine determinations using the rat prostate as a model tissue. Complete agreement between all five methods was obtained. Application of perchloric acid to formaldehyde-fixed sections of rat prostate strongly reduced theo-phthalaldehyde inducible and formaldehyde-fluorescamine inducible fluorescence characteristic of spermidine and spermine. Perchloric acid extracted 40% of tissue-bound polyamines from formaldehyde-fixed tissue sections, and molecules with the physicochemical characteristics of polyamines constituted 80–90% of all fluorescamine reactive molecules extracted. Our results therefore confirm the specificity of theo-phthalaldehyde and formaldehyde-fluorescamine methods for polyamine cytochemistry. As polyamines are strongly implicated in cellular growth regulation and cancer, simple and inexpensive techniques for polyamine histochemistry may be useful for interpreting the biological and pathophysiological roles of these molecules.     

Endogenous polyamines associate with DNA during its condensation in mammalian tissue. A fluorescence cytochemical and immunocytochemical study of polyamines in fetal rat liver

The polyamines spermidine and spermine are essential for cell proliferation and differentiation. By two independent fluorescence cytochemical methods as well as by immunocytochemistry, we have studied the distribution of these molecules in fetal rat liver. Strong reactions for polyamines were found in highly condensed chromatin, present in chromosomes in mitotic cells, and in condensed nuclei in late erythropoietic cells. Moreover, polyamines were so closely associated with DNA in condensed chromatin that DNase pretreatment was necessary for making them available for reaction with antibodies. In other cells, polyamines were mainly localized to the cytoplasm. Studies of cells at different stages in erythropoiesis revealed that polyamines become associated with DNA during its condensation and inactivation. Our data strongly indicate that polyamines participate in the condensation of DNA.     

Towards microfluorometric quantitation of polyamines in situ. Relationship between cellular polyamine concentration and fluorescence yield of the formaldehyde fluorescamine method

Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.     

Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.     

Localization and biosynthesis of polyamines in insulin-producing cells.

Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory granules in insulin-producing cells are discussed.     

Polyamines, molecules necessary for cell division, colocalize with peptide growth factors

The naturally occurring polyamines spermidine and spermine are necessary for cell division and growth. By restaining experiments, using three independent polyamine cytochemical methods, together with peptide immunocytochemistry, we show that substantial amounts of polyamines occur in a number of peptide growth factor-producing cell types. These include submandibular granular convoluted duct cells producing epidermal growth factor (EGF), pancreatic islet cells producing insulin and anterior pituitary cells producing growth hormone (GH). Other cell types in these tissues display only weak or no polyamine reactivity. Also blood platelets, known to contain platelet-derived growth factor (PDGF), are strongly stained for polyamines. Moreover, in EGF cells, insulin cells and blood platelets, polyamines are clearly localized in secretory granules. The possibility that polyamines may be coreleased and act in concert with peptide growth factors is discussed.     

Co-localization of a kallikrein-like serine protease (arginine esterase A) and atrial natriuretic peptide in rat atrium

Atrial natriuretic peptide (ANP) is stored in atrial granules primarily as a larger molecular weight precursor (pro-ANP), which is believed to be rapidly converted to an active peptide of 28 amino acids during or shortly after secretion. A tissue kallikrein-like serine protease has been suggested as a potential processing enzyme. In the present immunocytochemical study, using specific monoclonal antibodies, we found that esterase A, a kallikrein-like serine protease, was demonstrable in rat atrial myocytes and in ventricular myocytes, and was capable of cleaving pro-ANP to yield a low molecular weight product. Using colloidal gold immunocytochemistry at the electron microscopic level, we have found esterase A in atrial myocytes, both in granules and in another subcellular site that corresponds to sarcoplasmic reticulum. Double-label electron microscopic immunocytochemical results indicated that esterase A can co-localize with ANP in granules of atrial myocytes.     

Freeze-fracture cytochemistry: a new method combining immunocytochemistry and enzyme cytochemistry on replicas.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.     

Towards microfluorometric quantitation of polyamines in situ

Summary Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using -difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0–30 M) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.     

Digoxigenylated primary antibodies for sensitive dual-peroxidase labelling of neural markers

This study extends the application of the digoxigenin-anti-digoxigenin (DIG) technique to immunocytochemistry by using digoxigenin-tagged primary antibodies. Certain features of this technique when applied to non-radioactive in situ hybridization, such as the absence of endogeneous digoxigenin immunoreactivity in animal tissues, seem to be advantageous also for its application to immunocytochemistry. Thus, the present work is focused on dual-peroxidase staining experiments based on digoxigenylated antibodies directed against glial fibrillary acidic protein, parvalbumin, and calbindin, in a straightforward combination with conventional cytochemical methods. The protocols include the concomitant detection of two antigens, for which only primary antibodies from one animal species are available, with differently haptenized antibodies (e.g., biotinylated anticalbindin and digoxigenylated anti-parvalbumin). The versatility of the DIG technique is exemplified by the combination of lectin and immunocytochemical procedures for the detection of astrocytes and microglia, and the simultaneous visualization of perineuronal nets and parvalbumin-containing neurons in the rat brain.     

Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes

Recent immunocytochemical studies have shown that placental villous trophoblasts contain the high molecular weight cytokeratin (CK) proteins 5/6 and 17. In the case of CK 17, trophoblastic immunostaining was positive in villi covered by fibrinoid. CKs 5/6 and 17 are expressed by hyperproliferative cells. The aim of this investigation was to examine the location of these CKs in placental infarcts, known to be demarcated by fibrinoid and hyperproliferative trophoblasts. The results were compared with those obtained by immunostaining against Ki-67, tenascin and α1-, α6- and β1- integrins, which are involved in cell proliferation, differentiation and regenerative processes. Furthermore, the expression of the single CKs 7, 8, 10, 13, 14, 18 and 19 was investigated by immunocytochemistry and immunoblotting. While low and high molecular weight CKs were present in villous and extravillous trophoblasts, only low molecular weight CKs were detected in vascular and extravascular placental smooth muscle cells. Placental infarcts revealed different immunoreactivities in the infarct margin and centre: high molecular CKs, tenascin, Ki-67 and oncofoetal fibronectin predominated in the infarct margin, low molecular CKs, fibrin and integrins in the centre. The expression of tenascin and a defined change in the expression of CK 17 indicates villous repair and hyperproliferative mechanisms in placental infarcts. This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes

Recent immunocytochemical studies have shown that placental villous trophoblasts contain the high molecular weight cytokeratin (CK) proteins 5/6 and 17. In the case of CK 17, trophoblastic immunostaining was positive in villi covered by fibrinoid. CKs 5/6 and 17 are expressed by hyperproliferative cells. The aim of this investigation was to examine the location of these CKs in placental infarcts, known to be demarcated by fibrinoid and hyperproliferative trophoblasts. The results were compared with those obtained by immunostaining against Ki-67, tenascin and α1-, α6- and β1- integrins, which are involved in cell proliferation, differentiation and regenerative processes. Furthermore, the expression of the single CKs 7, 8, 10, 13, 14, 18 and 19 was investigated by immunocytochemistry and immunoblotting. While low and high molecular weight CKs were present in villous and extravillous trophoblasts, only low molecular weight CKs were detected in vascular and extravascular placental smooth muscle cells. Placental infarcts revealed different immunoreactivities in the infarct margin and centre: high molecular CKs, tenascin, Ki-67 and oncofoetal fibronectin predominated in the infarct margin, low molecular CKs, fibrin and integrins in the centre. The expression of tenascin and a defined change in the expression of CK 17 indicates villous repair and hyperproliferative mechanisms in placental infarcts. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cellular and subcellular localization of polyamines cytochemical methods providing new clues to polyamine function in normal and neoplastic cells

Summary Studies on the cellular and subcellular localization of polyamines have previously been hampered by the rapid redistribution of these organic cations during cell fractionation. Cytochemical methods selectively localizing polyamines would, hence, be important for our understanding of where polyamines are stored and where they work. With the aid of chemical characterizations of tissue extracts, biosynthesis experiments and cytochemical model experiments we show that the cytochemical formaldehyde-fluorescamine (FF) technique selectively detects the polyamines spermine and spermidine. Results obtained with the FF method concur with previous chemical observations by showing that growing and neoplastic cell populations are rich in cytochemically detectable polyamines. The FF method also localizes polyamines to certain normal secretory cell systems, including pancreatic exocrine and endocrine (insulin) cells, cells and secretion products in the ventral prostate, pituitary growth hormone cells and neurosecretory nerve endings and leucocytes. In most cell systems studied, polyamines appear to be most concentrated in cytoplasmic structures. However, in HeLa cell mitotic chromosomes and in frog red cell nuclei most polyamines seem to be associated with chromatin. These observations raise important questions concerning both extra- and intracellular functions of polyamines.     

FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy.

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for high-resolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.     

Microphotometric quantitation of the reaction product of several indirect immunoperoxidase methods demonstrating monoclonal antibody binding to antigens immobilized on nitrocellulose

The nitrocellulose model and microphotometry were used to investigate whether in immunoperoxidase cytochemical methods the amount of final reaction product reflects the amount of cell surface antigen. The results obtained with four cytochemical peroxidase methods, i.e., those using diaminobenzidine/H2O2 (DAB/H2O)2, DAB/H2O2/COCl2, DAB/H2O2/imidazole, and silver intensification of the DAB end product, were compared first. The quantitative DAB/H2O2/imidazole method proved to be the most sensitive and was selected for further studies. Cell surface antigens prepared by solubilization of peritoneal macrophages with octyl-beta-D-glucopyranoside were immobilized on nitrocellulose. Monoclonal antibody binding to these cell antigens was detected by peroxidase immunocytochemistry. Comparison of the sensitivity of the indirect immunoperoxidase and the biotin-(strept)avidin immunoperoxidase methods on the basis of the highest detectable dilution of a cell lysate showed that these methods were equally sensitive. A linear relationship between the absorbance of the peroxidase reaction product and the amount of cell lysate immobilized on nitrocellulose was found for all three indirect immunoperoxidase methods. This proves that the amount of final immunocytochemical peroxidase reaction product is proportional to the amount of antigen in cell lysates. However, the relative expression of antigens in intact cells differs from that in cell lysates. Therefore, the present method to solubilize cells and immobilize cell antigens cannot be used to quantitate the antigen content of cells.     

Immunocytochemical localization of a rhodopsin-like protein in the lipochondria in photosensitive neurons of Aplysia californica

Summary Polyclonal antibodies directed against squid opsin were used in immunocytochemical and immunoblot experiments to identify a rhodopsin-like protein in photosensitive neurons of Aplysia. Aldehyde-fixed abdominal and cerebral ganglia were embedded in paraffin for peroxidase anti-peroxidase analysis or used whole for immunofluorescence studies. Ganglia were embedded in Lowicryl K4M for electron-microscope immunocytochemistry. In both the cerebral and abdominal ganglia, light-microscope immunocytochemical results showed reaction product deposited around the neuronal cell periphery corresponding in position to the lipochondria. In the abdominal ganglion, the giant cell R2, located in the right rostral quarter, and neurons in the right caudal quarter were consistently labeled with anti-opsin. Electron-microscopic studies demonstrated ferritin-labeling of the lipochondria in R2 and other immunoreactive neurons. Immunoblot analysis of R2 and cerebral neuron extracts was used to identify two prominent immunoreactive protein bands at 85000 and 67500 molecular weight.