Localization of intermediate filament proteins in various structures of the human placenta as revealed by immunoperoxidase technique.

Formalin-fixed, paraffin embedded fragments of normal human placenta were subjected to immunoperoxidase technique with the use of Dako Kits appropriate for detection of vimentin, desmin, and cytokeratins (K 660, 530, and 518 respectively). Distribution of the intermediate filament proteins in syncytiotrophoblast, connective tissue of villous stroma, walls of blood vessels as well as in cells of basal plate and placental islets was compared. General pattern of staining and its localization was characteristic for each of the 3 types of antibodies. In spite of that, most structures observed revealed the presence of more than one type of intermediate filament proteins, with the exception of syncytiotrophoblast containing cytokeratins only. Staining of blood serum and of connective tissue matrix with anti-desmin and anti-cytokeratin antibodies was also observed, whereas fibrinoid remained always negative.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

Summary The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96%+1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin, Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde+ 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections as regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Rabbit peroxidase-antiperoxidase complex (PAP) as a model for the uptake of immunoglobulin G by the human placenta

Summary Rabbit peroxidase-antiperoxidase complex (PAP) has been shown to bind to IgG receptors on the human placental syncytiotrophoblast microvillar membrane. Its binding characteristics suggest that it is suitable as a probe for studies on the uptake of IgG by the human placenta.A novel assay system was developed to measure the dissociation constants (K _d) of the binding of PAP and of unlabelled human IgG to purified placental microvillar membranes. TheK _d for PAP was found to be 54 nM, while that for unlabelled IgG was found to be 17.5 nM.The uptake of PAP by placental tissue slices was observed using peroxidase histochemistry and electron microscopy. In initial experiments, reaction product was confined to the peripheral regions of the syncytiotrophoblast. Assaying a placental homogenate for catalase activity showed that it contained 250 units of activity per g wet weight of tissue (compared with 680 units/g for rat liver). Treatment of fixed tissue with the catalase inhibitor 3-amino-1, 2, 4-triazole allowed the localization of peroxidase reaction product in deeper regions of the syncytiotrophoblast. Based on observations of the localization of reaction product, we propose that PAP is taken up in coated pits, transferred into large apical multivesicular bodies, segregated into small vesicles which then transport it to the Golgi. From here the PAP is directed to the basal membrane by a mechanism as yet unknown.     

Demonstration of fibronectin in normal and acutely inflamed appendix

The presence and localization of fibronectin in normal and acutely inflamed appendices in man was studied using indirect immunoperoxidase technique on sections of formaldehyde fixed and paraffin embedded tissue, following pretreatment with pepsin and testicular hyaluronidase. In the normal appendix fibronectin was demonstrated in the region of the basement membrane of the surface epithelium, in the loose connective tissue, in the perimysium around the individual smooth muscle cells and in the vessel walls. In the acutely inflamed appendices, fibronectin was found in the luminal necrotic area, both intercellularly and in the cytoplasma of some inflammatory cells. In relation to the surface inflammation and in the tissue matrix corresponding to the acute inflammatory reaction fibronection was, compared to the normal appendix, found in increased amount. Furthermore, a comparison between the use of a primary antibody to fibronectin, produced in our collaborating laboratory and two different commercial primary antibodies showed that the staining results concerning the localization of fibronectin were equal for all three antibodies, whereas the commercial antibodies showed a weaker staining intensity and some unspecific staining compared to the antibody produced in our collaborating laboratory.     

Demonstration of fibronectin in normal and acutely inflamed appendix

Summary The presence and localization of fibronectin in normal and autely inflamed appendices in man was studied using indirect immunoperoxidase technique on sections of formaldehyde fixed and paraffin embedded tissue, following pretreatment with pepsin and testicular hyaluronidase. In the normal appendix fibronectin was demonstrated in the region of the basement membrane of the surface epithelium, in the loose connective tissue, in the perimysium around the individual smooth muscle cells and in the vessel walls. In the acutely inflamed appendices, fibronectin was found in the luminal necrotic area, both intercellularly and in the cytoplasma of some inflammatory cells. In relation to the surface inflammation and in the tissue matrix corresponding to the acute inflammatory reaction fibronection was, compared to the normal appendix, found in increased amount. Furthermore, a comparison between the use of a primary antibody to fibronectin, produced in our collaborating laboratory and two different commercial primary antibodies showed that the staining results concerning the localization of fibronectin were equal for all three antibodies, whereas the commercial antibodies showed a weaker staining intensity and some unspecific staining compared to the antibody produced in our collaborating laboratory.     

Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.     

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney

The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques. After preembedding immunostaining for laminin using IgG--PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A--PO, staining of the 1. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the 1. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the 1. fibroreticularis and the 1. rara but not in the 1. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.     

Increased immunohistochemical expression of thrombomodulin at placental perivascular myofibroblast in severe preeclampsia (PE)

The presence of pro-coagulant and anti-coagulant components of the placental vascular endothelium and syncytiotrophoblast are essential for homeostasis. Vascular endothelium prevents blood clot formation in vivo by involving a cell surface thrombin-binding glycoprotein, thrombomodulin (TM), that activates plasma anti-coagulant protein C. The TM levels increase during pregnancy, but the fibrinolytic capacity diminishes. Since vascular lesions with placental coagulation disorders can be associated with preeclampsia (PE), we hypothesized that TM expression in the stem villous vasculature and syncytiotrophoblast of the placenta are impaired in PE. Plasma and placental tissue samples were collected from PE (n=12) and normotensive pregnant patients (n=11). Patient's gestational age was 35.7+/-1.2 (normotensive) and 30.6+/-1.5 weeks (PE). Blood samples were drawn 30 min before delivery. Serum PAI-1 and PAI-2 antigens were determined by enzyme-linked immunoabsorbent assay (ELISA). A monoclonal antibody specific for TM was used for immunohistochemical tissue staining (ABC) and the staining was quantified by semi quantitative scores. Results show no intensity differences at the apical syncytiotrophoblast between the two groups. However, in preeclamptic placenta, TM expression diminished in the endothelium of the stem villi arteries and increased in the perivascular and stromal myofibroblats in cases of severe PE. TM changes were associated with an increased PAI-1/PAI-2 ratio. It is suggested that in severe PE, the decreased placental blood flow may be due to structural and functional impairment of the endothelium of the stem villi vessels and the surrounding perivascular and stromal myofibroblast, by increasing TM expression which may modulate fetal blow flow in the villous tree.     

Use of immunocytochemical techniques for the localization of human placental lactogen.

The recent claim by Gau and Chard (Br J Obstet Gynaecol 83:876, 1976) that, on theoretical grounds, it may be impossible to demonstrate the presence of human placental lactogen in placental tissue using the immunoperoxidase technique, has been reinvestigated. Placental tissue fragments fixed in Carnoy's fluid retained their morphologic identity compared with tissue fixed in formalin. Using these nonformalin fixed tissues, human placental lactogen was successfully localized within the cytoplasm of the syncytial layer of the placental villus. It is concluded that placental villi at term do in fact contain sufficient human placental lactogen to be demonstrated using the immunoperoxidase technique in contrast to the observation of Gau and Chard.     

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney

Summary The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques.After preembedding immunostaining for laminin using JgG-PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A-PO, staining of the l. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the l. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the l. fibroreticularis and the l. rara but not in the l. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.     

Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes: effect of different fixatives and of proteolytic digestion

The postembedding immunoperoxidase staining technique for the localization of immunoglobulins (light and heavy chains) and of lysozyme has been successfully applied to epoxy-embedded human lymph nodes, after removal of the resin. Glutaraldehyde-containing fixatives appear to be suitable for the immunohistochemical localization of human immunoglobulins and lysozyme, provided that the masked antigenicity of these proteins is recovered by proteolytic digestion of the tissue sections using 0.4% pepsin or 0.1% trypsin. Nonglutaraldehyde-containing fixatives allow the immunolocalization of human immunoglobulins without any enzymatic pretreatment. This study shows that tissues routinely fixed in glutaraldehyde and embedded for ultrastructural investigations are actually suitable for immunohistochemical studies on human immunoglobulins and lysozyme.     

Characterization of two monoclonal antibodies obtained after immunization with human liver mitochondrial membrane preparations.

Two monoclonal antibodies have been generated by fusion of mouse myeloma cells with spleen cells from mice immunized with human liver mitochondrial membranes. One antibody, 1H6/C12, an immunoglobulin G2a (IgG2a), binds to the inner membrane of rat hepatocyte mitochondria, and immunoperoxidase staining demonstrates that its epitope has an intracellular particulate distribution within rat and human hepatocytes and human brain neurons. The epitope reactive with 1H6/C12 is partially sensitive to proteinase digestion. The second antibody, 3F12/F2, an IgG1, binds to a contaminating cell type, namely the granulocyte, but it does not bind to monocytes, lymphocytes and red cells in human blood. This antibody reacts with cells in the portal tract and sinusoids of rat and human liver, as shown by immunoperoxidase staining. The epitope for 3F12/F2 is extremely sensitive to proteinase digestion and is only exposed when granulocytes are fixed in acetone, indicating an internal localization.     

Alkaline phosphatase activity in the placental labyrinth of the cat and problems of specific localization of transport adenosine triphosphatase. An ultrastructural study (author's transl)

The ultrastructural localizations of alkaline phosphatase (ALPase) and of Na+-K+-dependent adenosine triphosphatase (Na+-K+-ATPase) were studied in the placental labyrinth of the cat during the last days of gestation. ALPase activity could be detected in the syncytiotrophoblast but was absent from maternal tissues. Enzyme activity was observed only along plasma membranes of microvilli and absorption tubules on the maternal surface of the syncytium and also on the podocytes-like cytoplasmic processes of the fetal face. The localization of the Na+-K+-ATPase activity as obtained with the method of Ernst was identical with that of ALPase. This activity was not very ouabaine sensitive or K+ dependent, but was almost completely inhibited by levamisole. The strong ALPase activity of the syncytiotrophoblast does not allow a specific detection of Na+-K+-ATPase. However, the localization of these enzymes activities on syncytiotrophoblast surfaces directly related to fetal and maternal capillaries could suggest that these surfaces are associated with transport mechanisms of the trophoblast.     

Glypican-3 (GPC3) expression in human placenta: localization to the differentiated syncytiotrophoblast

The expression of glypican-3 (GPC3), a heparan-sulfate proteoglycan associated with the Simpson-Golabi-Behmel fetal overgrowth syndrome, was studied in normal human placental tissue and cell lines derived from human placentae. Cytotrophoblasts derived from term placentae expressed GPC3 mRNA at low levels in culture. GPC3 mRNA expression increased markedly during trophoblast differentiation. By contrast, fibroblast cell lines derived from normal placentae did not express GPC3 in culture. Similarly, choriocarcinoma cell lines derived from human placentae (BeWo, JAR, and JEG) failed to express GPC3 mRNA. In situ hybridization confirmed the localization of GPC3 mRNA to the syncytiotrophoblast. Furthermore, immunohistochemical staining of paraffin imbedded placental tissue demonstrated intense staining of the syncytiotrophoblast cell layer and less intense staining of cytotrophoblasts. No staining of mesenchymal elements was noted. These data confirm the presence of GPC3 in human placenta and suggest it is expressed by the differentiated syncytiotrophoblast at term.     

Characterization and localization of human placental ferritin.

Ferritin has been purified from normal full-term human placentae and its antigenic and molecular characteristics compared with adult liver ferritin. Placental ferritin is composed predominantly of a single subunit type, co-migrating with a liver ferritin standard on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Comparison of dose-response curves in an immunoradiometric assay indicated some tissue-specific antigenicity for placental ferritin. This was supported by immunofluorescence studies on cryostat sections of human placentae by using antibodies to placental and spleen ferritin. Specific staining for placental ferritin was demonstrated within placental syncytiotrophoblast, particularly localized towards the microvillus plasma membrane. Ferritin has also been shown by electrophoretic and antigenic analysis to be present in protein fractions solubilized from isolated human syncytiotrophoblast microvillus plasma-membrane preparations, suggesting that ferritin may play an active role in the transfer of iron from maternal transferrin across the syncytiotrophoblast plasma membrane.     

The value of hyaluronidase treatment of different tissues before demonstration of fibronectin by the indirect immunoperoxidase technique

The influence of testicular hyaluronidase treatment on the immunohistochemical localization of fibronectin in different tissues (human articular cartilage, large intestine, synovial membrane and experimental granulation tissue) as well on frozen as on formaldehyde fixed, paraffin embedded tissue, has been studied using the indirect immunoperoxidase technique. Pretreatment with hyaluronidase is essential in demonstrating fibronectin in frozen sections of human articular cartilage. In the other tissues examined treatment with hyaluronidase was not essential, but gave a more optimal staining quality. The effect of hyaluronidase treatment was to some extent dependent on the duration of treatment. In formaldehyde fixed, paraffin processed tissue the improvement with hyaluronidase treatment was only seen when the hyaluronidase followed pepsin digestion of the deparaffinized tissue sections.     

The value of hyaluronidase treatment of different tissues before demonstration of fibronectin by the indirect immunoperoxidase technique

Summary The influence of testicular hyaluronidase treatment on the immunohistochemical localization of fibronectin in different tissues (human articular cartilage, large intestine, synovial membrane and experimental granulation tissue) as well on frozen as on formaldehyde fixed, paraffin embedded tissue, has been studied using the indirect immunoperoxidase technique. Pretreatment with hyaluronidase is essential in demonstrating fibronectin in frozen sections of human articular cartilage. In the other tissues examined treatment with hyaluronidase was not essential, but gave a more optimal staining quality. The effect of hyaluronidase treatment was to some extent dependent on the duration of treatment. In formaldehyde fixed, paraffin processed tissue the improvement with hyaluronidase treatment was only seen when the hyaluronidase followed pepsin digestion of the deparaffinized tissue sections.     

Immunoelectron microscopic localization of aromatase in human placenta and ovary using microwave fixation

In this study we investigated the immunohistochemical localization of a unique aromatase, a single protein of 51,000 daltons, in the human placenta and ovary at light and electron microscopic levels. Microwave fixation was adopted for the immunoelectron microscopic study because it is an excellent method for preserving antigenicity and subcellular structures in frozen sections. Tissue samples from four immature human placentas, four full-term human placentas, and two human ovaries fixed in 10% formalin were examined by light microscopy. In addition, tissues from three full-term human placentas and one immature human placenta fixed in 4% paraformaldehyde were examined by electron microscopy. By light microscopy, immunoreactivity for this aromatase was located in the syncytiotrophoblast and a part of the cytotrophoblast of the placenta and in the lutein and granulosa cells of the ovary. Immunoelectron microscopy revealed that the aromatase antigen was localized on the surface of the microvilli, the lateral plasma membrane, and in the endoplasmic reticulum (ER) in the syncytiotrophoblast of the placenta. The positive immunoreactivity in the syncytiotrophoblast ER is consistent with previous results using antibodies for other types of aromatase, whereas the reactivity on the microvilli has not been previously described. The present report describes the fine localization of this unique aromatase in placental and ovarian tissues; its localization on the plasma membranes requires further physiological investigation.     

Demonstration of fibronectin in normal and injured aorta by an indirect immunoperoxidase technique

The presence and localization of fibronectin in normal and mechanically injured aorta in rabbits was studied using an indirect immunoperoxidase technique on tissue specimens fixed in formaldehyde, embedded in paraffin and pretreated with pepsin. The effect on staining quality of treatment with testicular hyaluronidase prior to immunoperoxidase staining was also examined. In the intima from normal aorta fibronectin was present in the subendothelial basal layer, along the internal and external elastic laminae, around and between the smooth muscle cells of the media and along the collagen and elastic fibres in the adventitia. Sixteen days after a single mechanical dilatation of the descending thoracic aorta all animals developed gross atherosclerotic-like changes. Microscopic examination revealed prominent neo-intimal hyperplasia with subendothelial, cushion-like thickenings but no medial or adventitial alterations. Fibronectin, in increased amounts, was found between and around the endothelial cells and in the subendothelial thickenings between the proliferating smooth muscle cells in relation to the fine, thin elastic and argyrophilic fibres. In the media and adventitia the amount and distribution of fibronectin was indistinguishable from uninjured control aortas. Treatment with testicular hyaluronidase before immunoperoxidase staining resulted in a higher staining resolution in normal and injured aorta. The conspicuous observation in the present study is that fibronectin exclusively accumulates in areas of tissue repair. The origins and functions of fibronectin during tissue injury and repair are discussed.