Versatility of anti-horseradish peroxidase antibody-gold complexes for cytochemistry and in situ hybridization: preparation and application of soluble complexes with streptavidin-peroxidase conjugates and biotinylated antibodies.

In previous studies we have employed a gold-labelled, affinity-purified polyclonal antibody against horseradish peroxidase (anti-HRP--gold) in the avidin-biotin peroxidase complex (ABC) technique and indirect labelled avidin-biotin methods. The gold-labelled antibody was used as final revealing reagent to replace the 3,3'-diaminobenzidine (DAB) reaction by immunogold silver staining. The anti-HRP--gold reagent proved to be advantageous since blocking of endogenous peroxidase activity in the tissue sections was not further required and staining of superior contrast and resolution could be achieved in paraffin sections. In the present study we have optimized this technique by combining the last two incubation steps, i.e. HRP-conjugated streptavidin and anti-HRP--gold. Different ratios of the two reagents were tested empirically to establish the conditions for the formation of a soluble complex with optimal staining properties. Quantitative evaluation by densitometry of the staining intensity showed that the soluble streptavidin-HRP/anti-HRP--gold complex and the indirect labelled avidin-biotin method employing the gold-labelled anti-HRP antibody performed equally well. Thus, the availability of this complex simplifies the streptavidin-biotin immunogold technique for immunohistochemistry, lectin histochemistry and in situ hybridization and further demonstrates the versatility of anti-HRP--gold complexes.     

Localization of calmodulin in flagella of zoospores ofPhytophthora cinnamomi

Summary Calmodulin distribution in the tinsel and whiplash flagella of zoospores ofPhytophthora cinnamomi has been studied by immunofluorescence microscopy and immunogold labelling. In whole zoospores labelled with a monoclonal antibody raised against pea calmodulin, followed by a second antibody-FITC, both flagella appear to be weakly stained except for a region at the base of the tinsel flagellum which was stained intensely. A similar staining pattern was also detected in isolated flagella labelled with anti-calmodulin. To identify the calmodulin rich region of the tinsel flagellum, we labelled sections of zoospores embedded in Lowicryl K4M with anti-calmodulin followed by a second antibody gold probe. In the tinsel flagellum, the gold labelling was restricted to a paraxonemal swelling close to the base. Very little gold labelling was detected elsewhere. The swelling extends for 1.5–2.0 n from the base of the tinsel flagellum and is hook shaped in cross section. Immunoblot analysis confirmed that the staining was specific for calmodulin.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

Summary A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H₂O₂ generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-feroprotein in liver and tissue polypeptide antigen in mainmary gland served as models. The immobilized twostep enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

Microwave-stimulated incubation in immunoelectron microscopy: A quantitative study

Summary Microwave irradiation has been applied to reduce the immunogold staining time of ultrathin sections of Lowicryl embedded specimens. Labelling has been stimulated by microwave irradiation during incubation with 10nm colloidal gold particls coated with either goat anti-mouse antibodies (GaM-gold) or goat anti-rabbit antibodies (GaR-gold) and has been compared with control incubations. Quantification has been performed on cytoplasmic membranes or lysosomes labelled with a primary antibody. Counting the gold particles over specific and non-specific sites in electron micrographs and electron microscopic images by IBAS 2000 revealed that irradiation of 25 l droplets both at 80W and 150 W resulted in an accelerated immunogold labelling, while the non-specific background levels were not increased. A plateau level in immunogold labelling intensity was reached after 25 min incubation under microwave irradiation at 150W as compared to 120 min incubation without microwaves. No improvement in localization sharpness of immunogold labelling on membranes was achieved by microwave irradiation. The microwave-mediated acceleration of immunogold staining may be considered as an example of a staining method with a restricted thermal action on microvolumes as indicated by direct temperature measurements using a fibre-optic thermometer.     

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues

Summary Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 12,400 of the primary antibody as compared to 1800 for the PAP technique.     

Immunocytochemical localization of β-1,3-glucan recognition protein in the silkworm,Bombyx mori

Summary A monospecific antibody against -1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.     

Sequential use of the PAP and immunogold staining methods for the light microscopical double staining of tissue antigens: Its application to the study of regulatory peptides in the gut

Double immunoperoxidase staining using different couplers can give various combinations of colours on a single tissue section to achieve a comparable picture of different antigens. However, the colour combinations achieved to date are not entirely satisfactory.A double immunostaining procedure is introduced here, combining the peroxidase anti-peroxidase (PAP) and immunogold staining (IGS) methods. The IGS method is a new, simple, sensitive and reliable approach to immunostaining at the light microscopic level. It was carried out in three ways. Firstly, a two-step method was used in which the second layer was goat anti-rabbit IgG adsorbed onto gold particles (GAR/Au20). Secondly, a three-step method was employed where the second layer was unlabelled goat anti-rabbit IgG and the third layer was a rabbit antibody to peroxidase adsorbed onto the gold particles (RAP/Au20) and acting as a gold-labelled IgG antigen. The third method combined the first two methods using GAR/Au20 as the second layer and RAP/Au20 as the third layer which increased the amount of bound gold and enhanced the red colour, providing a better picture.The use of gold-labelled antibodies in double immunostaining has great potential value for many studies including that of the diffuse neuroendocrine system of the gut.     

An alkaline-phosphatase staining method in avidin-biotin immunohistochemistry

An avidin-biotin alkaline-phosphatase (ABAP) staining method has been developed for the labeling of tissue sections and cell smears. The introduction of alkaline phosphatase as a marker enzyme through an avidin bridge results in excellent immunocytochemical labeling of different antigens using poly- and monoclonal antibodies. This technique avoids problems with endogenous peroxidase activity that sometimes occur using peroxidase staining procedures. The introduction of a preformed avidin-biotin alkaline-phosphatase complex (ABAPC) makes the presented technique as simple to handle as the widely used avidin biotin-peroxidase complex method (ABC). The ABAPC technique could be combined with other enzymatic labelings for double immunoenzymatic staining.     

Localization of collagen type VI in articular cartilage of young and adult mice

Collagen type VI was demonstrated immunomorphologically in articular cartilage (distal femur) of young (2–8 weeks) and adult mice by fluorescence and electron microscopy (gold-labelled second antibody—sandwich method) using pre-and post-embedding techniques. This collagen type was mainly seen in the vicinity of chondrocytes, and in larger amounts in adult cartilage. Electron-microscopic inspection (pre-embedding technique) revealed labelling above plaques that were 40–160 nm in size, and from which up to 7 fine filaments ( 10 nm) per unit sectional plane radiated. Using the post-embedding technique, only labelled plaques could be demonstrated; fine filaments were not perceptible. This was partly a result of the low contrast. It is assumed that the globular ends of up to 20 of the fine type VI filaments are anchored in one plaque and that the antibodies bind to the non-collagenous globular domains. Filaments radiated from the plaques and formed a threedimensional network that stabilized the structures of the cartilaginous matrix. Antibodies against fibronectin also labelled similar plaques. The ends of the type VI filaments are possibly linked into the plaques by fibronectin.     

Improved avidin-biotin-peroxidase complex (ABC) staining

Summary A considerable intensification of the avidin—biotin—peroxidase complex staining system (ABC) was obtained by sequentially overlaying the sections to be immunostained with an avidin-rich and a biotin-rich complex. Each sequential addition contributed to the deposition of horseradish peroxidase on the immunostained site and allowed the subsequent binding of a complementary complex. With this technique a higher dilution of the antisera could be used and minute amounts of antigen masked by the fixative could be demonstrated on paraffin sections.     

The localization of enzymes in tissue sections by immuno-histochemistry. Conventional antibody and mixed aggregation techniques

Synopsis Methods for detecting enzymes in tissue sections by antibody techniques are reviewed. In all these techniques, sections are first incubated with antibody. The bound antibody is visualized in one of four ways: identifying a label such as fluorescein linked to the antibody; using a labelled anti-antibody; employing complement and labelled anti-complement; or making use of a mixed aggregation immuno-cytochemical method.The last technique consists of three steps. A section is first incubated with antiserum, and secondly with the soluble enzyme under investigation. Thirdly the desired enzyme is stained using a conventional cytochemical method. The method is specific since, for example, the soluble enzyme used in the second step can bind only to antigenic determinants which are identical to those of the enzyme localized in the tissue. Thus purification of antigen and antibody sources is simplified, and chemical modifications of the antigen and antibody are avoided.Antibody also acts as a selective fixative for tissue antigen. It will inhibit the catalytic activity of its antigen and, in this way, permit the enzyme activity arising after the reaction of tissue enzyme-antibody complex with soluble enzyme to be amplified selectively. The mixed aggregation immuno-cytochemical technique has been used successfully with membrane-bound enzymes and cytoplasmic enzymes and for the demonstration of catalytically inactive enzyme precursors.     

The utility of photo-affinity labels as `mapping' reagents. A study of sub-populations of a specific rabbit antibody by using structurally related photo-affinity reagents

A specific rabbit antibody against the 4-azido-2-nitrophenyl determinant was photo-labelled by the homologous hapten ε-(4-azido-2-nitrophenyl)-l-lysine, and by the close structural isomer ε-(5-azido-2-nitrophenyl)-l-lysine. The extents of covalent labelling of the antibody-binding site were assessed by using radioactive haptens and exhaustive displacement dialysis, which leaves the unlabelled sites empty but largely intact. A single photolysis of hapten–antibody complex suffices to label those sites that are capable of being labelled. Although there is considerable overlap among sub-populations of antibody that will bind the two haptens non-covalently, sites that can be covalently labelled by one reagent cannot be labelled by the other.     

Immunocytochemical demonstration of the binding and internalization of growth hormone in GERL of Chang hepatoma cells

Summary The binding and internalization of endogenous growth hormone in Chang hepatoma cells were localized on the cell surface and in the Golgi-endoplasmic reticulum-lysosome (GERL) area by various indirect immunocytochemical labeling techniques, namely, peroxidase or colloidal gold conjugated to secondary antibody, and avidin-biotin complex methods. Rabbit antiserum and monoclonal antibodies raised against HPLC-purified porcine growth hormone were used in this study. In fixed material, antigen-antibody complexes were found to be homogeneously distributed along the cell membrane. Control groups showed negative binding on the cell surface. Trypsin treatment before immunolabeling removed antibody binding completely, but hyaluronidase was ineffective. Pretreatment of lectins did not block the recognition of primary antibody to antigen molecules on cell surface. Internalization of the antigen-antibody peroxidase or gold complexes was demonstrated in the cells, which were immunolabeled at 4°C, and then reincubated for 0–30 min at 37°C before fixation. After reincubation, the internalized ligand complexes were found in vesicles near the cell surface or in the GERL area near the Golgi apparatus which, however, did not label for peroxidase. These findings suggest that the trypsin-sensitive growth hormone, specifically bound and internalized into Chang hepatoma cells, is localized in the GERL instead of the Golgi apparatus and might be involved in the mechanism of tumor cell growth.     

Paired indirect immunoenzyme staining with primary antibodies from the same species. Application of horseradish peroxidase and alkaline phosphatase as sequential labels

Summary Paired indirect immunoenzyme staining based on primary antisera from the same species was performed sequentially without intermediate antibody elution. The first antigen was labelled brown by an immunoperoxidase procedure (either the two-stage indirect method, the unlabelled antibody peroxidase-antiperoxidase method, or the avidin-biotin bridge method using diaminobenzidine (DAB) and hydrogen peroxide as the substrates. The second antigen was labelled blue by applying a two-stage indirect immuno-alkaline phosphatase procedure using naphthol AS phosphate and Fast Blue BB salt as the substrate. In this way, polyclonal mucosal immunocytes were revealed in distinctly contrasting colours when stained for and light chains. Glucagon and somatostatin (D) cells in human pancreatic islets, and gastrin and D cells in human gastric antral glands, were likewise clearly differentiated. Conversely, a mixed colour appeared in some immunocytes after staining for and chains. However, unbalanced colour mixing was sometimes difficult to interpret, and additional experiments demonstrated that unwanted interactions could take place between the two sequences of reagents if the density of the DAB deposits was insufficient. These pitfalls were incompatible with unequivocal double staining in the same cell. Nevertheless, paired staining could be conveniently applied with the described procedures when prior knowledge had established that the antigens in question were located in separate cells.     

Demonstration of ricin within the mammalian para-aortic lymph node I. Comparison of the localization, after intramuscular injection, with three immunocytochemical methods

Summary Following a supralethal injection of ricin into thigh muscle of the adult rat, the toxin was demonstrated post-mortem in the para-aortic lymph node, ipsilateral to the side of injection. The relative merits of two immunoenzyme methods, peroxidase anti-peroxidase (PAP) and avidin—biotin—peroxidase complex (ABC) and a silver-enhanced immunogold method (IGSS) were assessed in the detection of ricin in the lymph node tissue. The toxin was clearly seen to be located in association with histiocytes found both within and lining the sinuses of the nodes and also, in some cases, in the subcapsular sinus of the node; the toxin was not demonstrable within lymphoid follicles by light microscopy. However, using electron microscopy and the IGSS technique, cells carrying discrete particles of gold could be visualized within follicular areas. The IGSS and ABC-peroxidase methods were both found to give excellent results without background staining at the light microscopy level. However, when these techniques were used prior to embedding and viewing by electron microscopy, the IGSS technique proved to be far superior.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H2O2 generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3'-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-fetoprotein in liver and tissue polypeptide antigen in mammary gland served as models. The immobilized two-step enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

The ER-TR4 monoclonal antibody recognizes murine thymic epithelial cells (Type 1) and inhibits their capacity to interact with immature thymocytes: immuno-electron microscopic and functional studies

The thymic stroma is heterogeneous with regard to cellular morphology and cellular function. In this study, we employed the monoclonal antibody ER-TR4 to characterize stromal cells at the ultrastructural level. To identify the labelled cell type, we used two techniques: immunogold labelling on ultrathin frozen sections and immunoperoxidase staining on thick vibratome sections. ER-TR4 reacted with thymic Type 1 epithelial cells (according to our classification). A dense labelling appears in the cytoplasm of cortical cells using the two techniques. Immunogold labelling identified small cytoplasmic vesicles whereas the cytoplasm and the cell membrane seem to be labelled with the immunoperoxidase technique. ER-TR4 also identified isolated thymic nurse cells (TNC), and was observed in vitro to inhibit the capacity of some type 1 epithelial cells to establish interactions with immature thymocytes. This finding supports the hypothesis that the factor is involved in the formation of lymphoepithelial interactions within thymic nurse cells, and thus in the relations that immature thymocytes establish with the thymic microenvironment.     

Electron-microscopic demonstration of α-tubulin immunoreactivity in astroglia

Summary Vibratome sections of the cerebral cortex, hippocampus and cerebellum were immunostained for -tubulin using the TU-Ol monoclonal antibody. In all three regions, electron microscopy of the immunostained preparations revealed — in addition to the previously described reaction of pyramidal apical dendritic microtubules — consistent staining of the ribosomal apparatus of astrocytes.     

Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat

Summary The histological localisation of -D-galactopyranosyl residues in glycoconjugates of rat stomach and duodenal mucosae was studied by use of Griffonia simplicifolia agglutinin I, i.e. the isolectin mixture (A+B) and the isolectin B₄ (B₄). Cryostat sections which were either unfixed or acetone fixed and paraffin sections from both ethanolacetic acid and formaldehyde fixed tissue blocks were compared. Cellular details were better preserved in paraffin than in cryostat sections. Reactivity of cells binding GS I was less sensitive after formaldehyde than after ethanol-acetic acid fixation inasmuch as higher concentrations of lectins were needed. This drawback could be overcome by trypsinisation of the sections. The binding pattern of GS I (A+B) corresponded with that of GS I (B₄) in either cryostat or paraffin sections. GS I was detected in the cytoplasm of parietal cells and in Brunner's gland cells. In duodenal crypts and villi, lectin was bound to supranuclear regions in the cytoplasm of columnar and goblet cells. The staining efficiency of fluorescein (FITC), horseradish peroxidase (HRP) and colloidal gold particle (CGP) labels in both direct and indirect lectin stainings was compared. Under all experimental conditions, indirect methods required lower concentrations of lectins than direct ones; indirect procedures increased sensitivity about 5–10 fold. CGP labels were always of highest sensitivity when gold particles were further developed by a silver precipitation method. HRP was not as efficient in lectin localisation as CGP, but cytochemical staining was more convenient in routine work. Direct FITC labellings proved to be of lowest sensitivity.     

Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures

The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the several staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotin-binding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. During formation of the complex, avidin acts as a bridge between biotin-labeled peroxidase molecules; and biotin-labeled peroxidase molecules, which contains several biotin moieties, serve as a link between the avidin molecules. Consequently, a "lattice" complex containing several peroxidase molecules is likely formed. Binding of this complex to the biotin moieties associated with secondary antibody results in a high staining intensity.