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.     

Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method

We describe a simple and effective method for inhibition of endogenous peroxidase activity in the immunoperoxidase technique. Specimens are pre-treated with a mixture of azide and hydrogen peroxide, which is then followed by an indirect immunoperoxidase procedure. Comparison studies showed no significant loss of antigenicity or morphological details by this pre-treatment. The method is most useful for evaluating cell-specific antigens on specimens that have abundant endogenous peroxidase activity, such as blood and bone marrow.     

Detection of herpes simplex virus infection using glucose oxidase-antiglucose oxidase immunoenzymatic stain

Herpes simplex virus (HSV) infected cells have been detected in tissue culture and human cell specimens by an immunoenzymatic staining method using the fungal enzyme glucose oxidase. Infected cells from culture or human specimens appear as dark blue, brown, or red, depending on the tetrazolium salt used in the disclosing reaction, with virtually no staining of uninfected cells. The specificity and sensitivity of this method and of the more commonly used immunoperoxidase method are comparable, but the immunoglucose oxidase method avoids the problems of nonspecific staining by the endogenous peroxidase present in mucosecretions and inflammatory cells. Staining time can be reduced up to 40% of that necessary for the unlabeled immunoperoxidase procedure without compromising the quality of staining results.     

Characterization of the population of phagocytic cells in thymic cell suspensions

Rat thymic phagocytic cells were characterized in vitro using various light- and electron-microscopical techniques. Thymic cell suspensions were mechanically prepared and enriched for non-lymphoid cells, which were predominantly phagocytic and of three types. Type I showed acid phosphatase (APh) activity in small granules dispersed throughout the cytoplasm and were mostly Ia antigen-positive, although the Ia membrane label varied in intensity and distribution among individual cells. Only a few cells had endogenous peroxidase activity. The type-I cells could not be clearly distinguished morphologically from type-II or -III cells, and most likely comprise precursors of both these cell types. Type-II were large pale cells with many slender cell processes. These cells had APh activity centrally positioned, were strongly positive for Ia on the cell membrane and were negative for endogenous peroxidase. The cytoplasm frequently contained Birbeck granules, which unequivocally classifies these cells as the in vitro equivalent of the interdigitating cells present in the medullary area of the thymus in situ. Type-III cells were rounded with a smooth or ruffled cell membrane and contained vacuoles and many phagolysosomes. They were strongly positive for APh which was present throughout the cytoplasm. About 50% of these cells were positive for endogenous peroxidase in a pattern resembling resident macrophages. The cells were negative for Ia antigens. Type-III cells mostly likely represent the macrophages found in the cortical area of the thymus.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes non-specific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidin-biotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

Summary The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes nonspecific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidinbiotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Simultaneous visualization of two antigens in the same tissue section by combining immunoperoxidase with immunofluorescence techniques.

A simple method was developed whereby immunoperoxidase and immunofluorescence techniques were applied in consecutive steps to demonstrate the presence of two antigens in the same tissue section. This method was applied in three model, two antigens were shown: a) each (gastrin and pepsinogen II) inside one of two different cell types (gastrin (G) and antral peptic cells), b) each (kappa or gamma light chains) inside different cells of the same type (plasma cells); also, both (kamma and gamma light chains) inside the same cell (Reed-Sternberg cell), and c) both (pepsinogen I and II) inside the same cell (chief cell of oxyntic glands). The results could be viewed and photographed either simultaneously, when the antigens were in different cells, or sequentially, when the antigens were in the same cells.     

The development of the resident pattern of endogenous peroxidatic activity in mouse peritoneal macrophages coincides with the expression of the differentiation antigen F4/80. A combined method for immunoperoxidase labeling and endogenous peroxidase cytochemistry

In the mouse the maturation of mononuclear phagocytes was followed by comparing the ultrastructural pattern of endogenous peroxidatic activity (PA) at different time points during an acute peritonitis induced with newborn calf serum (NCS). Exudate macrophages demonstrate PA only in lysosomes, whereas resident macrophages have reaction product in the nuclear envelope (NE) and rough endoplasmic reticulum (RER). Transitional cells called "exudate-resident" macrophages have PA in the NE, RER, and some virginal lysosomes. In addition, peroxidase-negative macrophages were also present. A monoclonal antibody, F4/80, that specifically recognizes a mouse macrophage differentiation antigen (Austyn and Gordon, 1981) was used in this study. To compare the indirect immunoperoxidase labeling of this antigen and the endogenous peroxidase cytochemistry on the cellular level, a combined method was developed. Finally, the method was applied to the peritoneal cells at different time points after intraperitoneal injection of NCS in mice. The relative numbers of cells demonstrating the different patterns of endogenous PA and the proportions of each subpopulation expressing F4/80 antigen were estimated. It appeared that the expression of the antigen F4/80 coincides with the development of the resident pattern of PA. It is therefore concluded that the macrophages with the resident pattern of endogenous peroxidase are derived from monocyte-like exudate macrophages. In addition, the results indicate that both exudate-resident macrophages and at least a part of the peroxidase-negative macrophages are transitional forms.     

Resolution of diaminobenzidine for the detection of horseradish peroxidase on surfaces of cultured cells

The resolution of indirect immunoperoxidase methods for localizing antigens on the surface of plasma membranes of cultured cells was tested using dissociated monolayer cultures of ciliary ganglion neurons prelabeled with cationic ferritin. Clusters of ferritin were produced on the cell surface by warming the cells to 37 degrees C after the ferritin, rabbit anti-ferritin, and goat anti-rabbit immunoglobulin coupled to horseradish peroxidase had all been applied. Intense 3,3'-diaminobenzidine tetrahydrochloride (DAB) staining was limited to the regions immediately surrounding the ferritin clusters. The lateral spread of the DAB reaction product beyond the outer ferritin particles in each cluster averaged 54-81 nm in four experiments. A second type of increased density, coinciding with the thickness of the plasma membrane, was also seen. These stained plasma membranes extended 161-339 nm from the ferritin clusters.     

Non-deleterious inhibition of endogenous peroxidase activity (EPA) by cyclopropanone hydrate: a definitive approach

Endogenous peroxidase activity (EPA) poses a serious problem in immunoperoxidase localization of antigens unable to withstand deleterious effects of aldehyde fixatives, alcohols, and various oxidative reagents. This has forced the development of more selective inhibition methods. Of these, phenylhydrazine or azide combined with small amounts of H2O2 have proved quite effective. However, the precise mechanism of the action of these compounds on EPA generating proteins is not understood. Cyclopropanone hydrate is a compound whose inhibitory action on the heme moiety of horseradish peroxidase is well understood. The aim of this study was to investigate the effect of this compound on EPA and to compare its efficiency with that of optimal phenylhydrazine and sodium azide regimens. In addition, any gross deleteriousness of cyclopropanone hydrate towards immunoperoxidase immunolocalization of three of the most delicate lymphocyte surface antigens was investigated. Cyclopropanone hydrate was found to inhibit EPA with progressing strength between 0.15-15 mM. Over this range, H2O2 was found necessary for inhibition only for cyclopropanone hydrate concentrations up to 0.15 mM. Beyond this amount, the compound inhibited EPA equally strongly in the presence or absence of H2O2, reaching near-maximum inhibition at 15 mM. This and the H2O2-requiring regimens were found to cause no gross diminution in immunoperoxidase staining of CD4, CD6, and CD8 antigens in snap-frozen, acetone-fixed human tonsil sections. Cyclopropanone hydrate therefore provides a definitive non-deleterious mode of inhibiting EPA for immunoperoxidase staining of delicate antigens.     

Immunocytochemistry of cerebrospinal fluid

In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined. Immunocytochemical studies could be performed on either cell suspensions or smears. The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears. Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity. The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique. The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining. Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears. In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method. These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses.     

Endogenous phosphatase activity in tumor cells in a liver aspirate. A potential problem for immunocytodiagnosis using immunoalkaline phosphatase methods

Immunocytochemical studies were performed on fine needle aspirates of the liver in a patient with hepatocellular carcinoma. A panel of commercially available antibodies was used to study the aspirated cells by immunoalkaline phosphatase and immunoperoxidase methods. The malignant cells in the aspirates, which were positively stained by the immunoperoxidase method for alphafetoprotein and by both methods for epithelial membrane antigen, were most probably hepatocellular in origin. Some cells were shown by the immunoalkaline phosphatase method to possess leukocyte-common antigen (LCA) and antigens of colonic and ovarian tissues. These findings were further investigated, and it was found that the tumor cells indeed had LCA as well as levamisole-resistant alkaline phosphatase activity. Although the immunoalkaline phosphatase methods are useful immunodiagnostic techniques applicable to fine needle aspirates, the endogenous enzyme activity present in some nonhematopoietic tumor cells is a cause for caution in the use of these methods in aspirates from nonhematopoietic tumor tissues.     

Immunohistochemical detection of mammary tumor virus antigens in sweat and sebaceous glands of mice

Mammary tumor virus (MTV) antigens in both sexes of GRS/A, SHN and C3H mice were examined in the sweat and sebaceous glands by immunoperoxidase technique using antiserum against gp52, envelope protein, or p27, core protein. Balb/c mice were used for reciprocal foster nursing with these inbreds to discriminate the expression of endogenous MTV from that of exogenous MTV. Both antigens were first detected around the age of 4 months in the sweat glands of mice with endogenous GR- or SHN- MTV. A linear staining of gp52 was seen along the luminal borders of glandular cells, and the reaction products for gp52 were demonstrated on the apical cell membranes, where no virion could be seen ultrastructurally. A diffuse staining of p27 was found in the cytoplasm of some glandular cells, where MTV particles could not be detected. In the sebaceous gland of the same mice, however, only p27 was first detected at the age of 60 days. A dot-like staining of p27 was found in the perinuclear region of some glandular cells, where an aggregation of intracytoplasmic A particles could be seen under an electron microscope. These positive stainings were unrelated to sex. In such skin appendages of all examined C3H mice and Balb/c mice with GR- or SHN- MTV, no antigen expression could be seen up to the age of 500 days. Therefore, some genes might be able to regulate the expression of endogenous MTV antigens in the skin appendages, while their glandular cells would have no receptor for exogenous MTV, namely the so-called "milk factor".     

Application of the avidin-biotin-complex method for the light microscopic analysis of lymphocyte subsets with monoclonal antibodies on air-dried smears

A study was undertaken of the application of the avidin-biotin-peroxidase complex (ABC) method to the monoclonal antibody MAbs staining of mononuclear cells in hematologic and cytodiagnostic materials. Satisfactory cell morphology and immunoreactivity of surface antigens were observed when the slides were fixed in 80% acetone in phosphate-buffered saline or in 60% acetone in 0.03 M citric acid buffer solution (pH 5.4). Unstained air-dried preparations could be preserved for two weeks at room temperature in a desiccator and for one year at -70 degrees C after fixation. An excellent immunoreaction, even with a weak surface antigen, was observed by inhibition of endogenous peroxidase after the secondary antibody reaction; reactions of weak antigens tended to be obscured when the inhibition was performed before the first antibody reaction. Use of the Giemsa stain as a counterstain made it possible to readily observe the cell morphology; therefore, white blood cell analysis could be performed simultaneously when peripheral blood smears were studied. The positive rate of immunoreaction by an immunofluorescent method was well correlated with that obtained by the ABC method. The ABC method proved to be an excellent immunocytochemical technique for detecting cell surface antigens with high sensitivity and specificity; furthermore, it is useful for cell morphology studies and yields permanent preparations.     

Immunoelectron microscopic labeling of immunoglobulin in plasma cells after osmium fixation and epoxy embedding

Previous studies have found that immunoglobulin cannot be immunolabeled in tissues prepared for electron microscopy by usual methods. To test this conclusion, we used a protein A-gold postembedding immunolabeling method on tissues that were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epoxy resin; sections were pretreated with sodium metaperiodate. A variety of common fixation protocols were also used and the most suitable conditions for immunolabeling were determined. This technique permitted the ultrastructural localization of immunoglobulin light chains in optimally preserved and contrasted plasma cells from human tonsil, lymph nodes, plasmacytomas, and a renal biopsy. We were able to demonstrate multiple antigens in the same tissue and label antigens in tissues that had been stored for many years in epoxy resin. The technique allows quantitation of the gold label over plasma cell organelles and therefore gives information about the immunoglobulin secretory pathway in these cells. We found that the protein A-gold procedure compares favorably in technical ease with the immunoperoxidase, avidin-biotin peroxidase, and immunoglobulin-colloidal gold immunolabeling methods, and has added advantages in allowing precise localization and quantitation of the labeled antigen.     

Immunocytochemical characterization of S-100 beta-positive human T-lymphocytes by a double immunostaining method

The antigenic properties of S-100 beta-positive human T-lymphocytes (S-100 beta+ T-cells) were investigated by a double immunostaining technique employing an indirect immunoperoxidase method for cytoplasmic S-100 beta subunit and an immunoalkaline phosphatase method for cell surface antigens detected by various monoclonal antibodies to human lymphocytes. S-100 beta+ T-cells recognized by their diffuse intracytoplasmic immunoperoxidase reaction, also expressed CD2, CD3, CD8 antigens demonstrated by surface blue alkaline phosphatase reactivity, but not CD4, CD1, CD25 (interleukin-2 receptor), or HLA-DR antigens. However, they displayed a blastic change to T-cell mitogens, such as Concanavalin A(Con-A) and PHA, followed by the expression of CD25 and HLA-DR antigens. Under normal conditions, S-100 beta+ T-cells comprised approximately 5-22.8% of CD8+ cells amongst human peripheral blood mononuclear cells.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Localization and quantification of hormones, ligands, and mRNA with affinity-gold probes

The application of immunogold techniques to localize pituitary hormones produces label that can be quantified and correlated with different secretory states. This report focuses on three major applications of the technology. In the first set of studies, immunogold labels for adrenocorticotropin (ACTH) or luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) were applied to ultrathin sections of pituitaries from adrenalectomized rats or from rats in different stages of the estrous cycle. During the first week after adrenalectomy, ACTH cell area increased. The concentration of immunoperoxidase label (amount of label/area of the corticotropes) decreased. Counts of gold markers showed that there were no changes in the concentration of antigens per granule. Three weeks after adrenalectomy, the amount of immunoperoxidase label increased along with the concentration of that label. The concentration of gold label for ACTH on granules also increased. All changes correlated well with increases in serum ACTH stimulated by adrenalectomy. In the studies of cycling rats, gonadotropes showed increases in the number of gold markers for LH beta or FSH beta per granule area just before an elevation in serum levels. There were also increases in the proportion of granules that contained only LH beta or FSH beta (monohormonal) before the rise in secretion. Thus, nonparallel release of gonadotropins might be attributed to changes in the ratio of gonadotropins packaged per granule. In the second series of studies, avidin-gold labels were used to identify sites of binding of biotinylated ligands. These studies illustrate and quantify binding by biotinylated gonadotropin-releasing hormone (GnRH) to ovarian or pituitary target cells. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show that the target cells in the pituitary contain gonadotropins. In the third set of studies, avidin gold or avidin peroxidase was used to label sites of hybridization of a biotinylated cRNA probe to gonadotropin beta subunit mRNA. The sites of hybridization appear on rough endoplasmic reticulum; however, further work is needed to improve cell ultrastructure and perserve antigens. Triple-labeling protocols (avidin-peroxidase followed by immunogold) show the feasibility of the technique as well as the need for further refinement. To summarize, these studies describe multiple applications of gold labels for the localization of antigens, ligands, and mRNA. The labels are sensitive for detection of antigens and ligands and easily quantified. Quantitative analyses show changes in concentration of gold label that correlate well with secretory states.     

Elastase and cathepsin G of human monocytes: heterogeneity and subcellular localization to peroxidase-positive granules

We used antibodies to human leukocyte ("neutrophil") elastase and cathepsin G to localize the corresponding antigens in human neutrophils, monocytes, and alveolar macrophages by immunohistochemistry. Furthermore, we combined immunogold localization with enzyme histochemistry to localize proteinase antigens and endogenous peroxidase activity in the same sections. As expected, all neutrophils contained both elastase and cathepsin G, and the proteinases localized to granules with peroxidase activity. In contrast, marked heterogeneity in monocyte staining for elastase, cathepsin G, and endogenous peroxidase was found. Sixty percent or more were unstained, while the remainder varied greatly in staining intensity. The elastase and cathepsin G in monocytes were localized by immunoelectron microscopy, combined with histochemistry, to cytoplasmic granules which had peroxidase activity. Alveolar macrophages were unstained. Therefore, a subpopulation of peripheral blood monocytes contains leukocyte elastase and cathepsin G in a cell compartment from which these enzymes may potentially be released into the extracellular space. The occurrence of peroxidase and neutral proteinases in the same granules in monocytes could permit the H2O2-myeloperoxidase-halide system and the neutral proteinases to act in concert in such functions as microbe killing and extracellular proteolysis.