Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors.

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.     

A specific and sensitive double-immunofluorescence method for the demonstration of S-antigen and serotonin in trout and rat pinealocytes by means of primary antibodies from the same donor species

 Immunocytochemical double-labeling methods are important tools in cell and neurobiology. Here we describe a method which is based on double immunofluorescence and allows specific detection of two different antigens located in the same cell compartment by two primary antibodies raised in the same species. As an example, we present the double-immunolabeling method for the S-antigen (SAg), a photoreceptor-specific protein, and the indoleamine serotonin (5HT) in dissociated trout and rat pineal cells immobilized on coversliped and in frozen sections of the trout pineal organ. As a first step, the preparations on the slides or coverslips were sequentially incubated with the first primary antibody (rabbit anti-SAg), the fluorescein-labeled (anti-rabbit) secondary antibody, and then with normal rabbit serum. Meanwhile, the second primary antibody (rabbit anti-5HT) was coupled to a Cy3-labeled secondary (anti-rabbit) antibody in a reaction tube and excess binding sites were quenched with normal rabbit serum. This complex was applied to the specimens after completion of the first (SAg) immunoreaction on the slide. For control experiments, the first (anti-SAg) or the second (anti-5HT) primary antibody were omitted. Most of the rat and trout pinealocytes were double immunolabeled for SAg and 5HT. In the trout, few cells contained SAg or 5HT immunoreaction only. This underlines the selectivity of each immunoreaction. The results show that the method can be used for the analysis of whole cells and tissue sections by means of conventional fluorescence and confocal laser scanning microscopy. Accepted: 20 October 1997     

Application of antigen retrieval by heating for double-label fluorescent immunohistochemistry with identical species-derived primary antibodies.

Double-label fluorescent immunohistochemistry (IHC) is frequently used to identify cellular and subcellular co-localization of independent antigens. In general, primary antibodies for double labeling should be derived from independent species. However, such convenient pairs of antibodies are not always available. To overcome this problem, several methods for double labeling with primary antibodies from identical species have been proposed. Among them are methods using monovalent secondary antibodies, such as Fab fragments. Soluble immune complexes consisting of primary and monovalent secondary antibodies are first formed. After absorption of the excess secondary antibody with nonspecific immunoglobulin, the immune complexes are applied to sections. By this procedure, unwanted cross-reaction between false pairs of antibodies is avoidable. However, soluble immune complexes often show reduced or no immunoreactivity to antigens on sections. I noted that antigen retrieval (AR) of tissues by heating often but not always showed improved immunoreactivity for soluble immune complexes. Here I demonstrate the examination of conditions for this soluble immune complex method using AR-treated sections and show examples of double-label fluorescent IHC with identical species-derived primary antibodies.     

Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies

Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein–conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies. (J Histochem Cytochem 56:969–975, 2008)     

Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.     

Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid, choline acetyltransferase or serotonin

In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gamma-amino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H2O2 (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H2O2 (brown reaction product in target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.     

免疫酶双重染色中抗体洗脱的进一步研究

本实验进一步检查了三种常用的洗脱方法从切片上除去免疫酶组织化学染色后的抗体的效果。结果表明,PAP法染色后,氧化法的抗体洗脱完全,效果可靠;酸洗法和酸洗/二甲基甲酰胺法的效果视不同抗体而不同,二甲基甲酰胺单用或用于酸洗后似无效。所用方法均不能从ABC法染色的垂体组织切片上完全除去抗体复合物。因之,将ABC法用于第一抗体来源于同一动物种的双重染色中,来显示第一种抗原时,要特别注意排除假性双标记。     

Chromogen-based immunohistochemical method for elucidation of the coexpression of two antigens using antibodies from the same species

The authors established a chromogen-based, double immunolabeling method using antibodies from the same species without any unwanted cross-reactivity. In addition, time-consuming staining steps were shortened by using polymer-based secondary antibodies. Taking advantage of the nature of the chromogen 3-amino-9-ethylcarbazole (AEC), which is used as a horseradish peroxidase substrate for antibody detection, the AEC-derived signals in the first color development were easily eliminated by alcohol treatment. Therefore, the signals from the first staining did not interfere with those from the subsequent second staining, which used the chromogen 3,3'-diaminobenzidine. The co-localization of antigens within the same cell could be confirmed using this method, because cell images of the individual dye staining steps could be obtained and developed. The images from each step could be expressed in pseudo-colors in a dark field by using a computer. As a result, merged images could be constructed that resembled the images acquired by the fluorescent immunolabeling technique. The resolution of this method enabled analysis of the coexpression of two antigens in the same cell in the same section. The authors have named this staining technique the elucidation of the coexpression of two antigens in a cell using antibodies from the same species (ECSS).     

Combined use of immunoperoxidase and radioimmunocytochemistry for double immunocytochemical labeling of neurons at light and electron microscopic level

Complexes formed by binding 125I- or 3H-labeled neuropeptides to one of the two binding sites of their specific antibodies allowed specific and sensitive labeling of various peptidergic neurons, which could be detected by classical autoradiographic methods. To visualize two neuronal antigens on the same material at both light and electron microscopic level, we used a new technique of double immunocytochemical labeling, combining immunoperoxidase and radioimmunocytochemistry. The main steps of the process included: (a) indirect labeling of the first antigen by its specific antibody and by a peroxidase-labeled Fab immunoglobulin fragment directed against the primary antibody; (b) direct labeling of the second antigen by a radiolabeled peptide-antibody complex; (c) revealing of the first label in the presence of peroxidase substrate; and (d) revealing of the second label by autoradiographic treatment of tissue sections. Compared with other known techniques of double immunostaining, this technique offers major advantages for combined visualization of two neuronal antigens at the electron microscopic level: (a) two neuron types can be labeled by a pre-embedding approach, allowing highly sensitive detection of neuronal antigens throughout the 50-microns thickness of vibratome sections; (b) two primary antibodies obtained in the same species can be used to label the two antigens without any risk of crossreactions between the two successive labelings; and (c) the two labels can easily be differentiated, even when they are co-localized within the same neuron structures. Application of this double immunostaining technique is illustrated by data obtained in rat hypothalamus concerning the relationships among a variety of identified neurons and the co-localization of different neuropeptides within the same neuron system.     

Tyramide Signal Amplification Method in Multiple-Label Immunofluorescence Confocal Microscopy

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.     

Antibody arrays for high-throughput screening of antibody-antigen interactions

We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.     

Recent advances in the detection and characterization of specific antibody-forming cells in tissue sections

Summary A new general approach has been developed for the detection of one or more different specific antibody producing cells and the simultaneous determination of their Ig isotype in tissue sections, after immunization of animals. Specificity of intracellular antibodies is demonstrated after incubation of the sections with an antigen-enzyme conjugate and the isotype of the antibodies is determined using an anti-immunoglobulin (Fc chain-specific)-enzyme conjugate followed by histochemical revelation of the two different enzymes. The principles of the method, the required antigen— and antibody—enzyme conjugates and their application in single, double or triple staining studies are reviewed.The method allows the detection of specific antibody-forming cells against protein antigens as well as against haptens. By means of haptens such as trinitrophenyl (TNP), immune responses against thymus dependent, thymus independent, and particulate antigens can be studied. In a limited number of cases the method can also be used to study the localization of antigen—antibody complexes.     

Screening one bead one compound libraries against serum using a flow cytometer: Determination of the minimum antibody concentration required for ligand discovery

One bead one compound (OBOC) libraries can be screened against serum samples to identify ligands to antibodies in this mixture. In this protocol, hit beads are identified by staining with a fluorescent labeled secondary antibody. When screens are conducted against two different sets of serum, antibodies, and ligands to them, can be discovered that distinguish the two populations. The application of DNA-encoding technology to OBOC libraries has allowed the use of 10?µm beads for library preparation and screening, which pass through a standard flow cytometer, allowing the fluorescent hit beads to be separated from beads displaying non-ligands easily. An important issue in using this approach for the discovery of antibody biomarkers is its analytical sensitivity. In other words, how abundant must an IgG be to allow it to be pulled out of serum in an unbiased screen using a flow cytometer? We report here a model study in which monoclonal antibodies with known ligands of varying affinities are doped into serum. We find that for antibody ligands typical of what one isolates from an unbiased combinatorial library, the target antibody must be present at 10–50?nM. True antigens, which bind with significantly higher affinity, can detect much less abundant serum antibodies.     

Conformational changes in a Vernier zone region: Implications for antibody dual specificity

Understanding the determinants of antibody specificity is one of the challenging tasks in antibody development. Monospecific antibodies are still dominant in approved antibody therapeutics but there is a significant body of work to show that multispecific antibodies can increase the overall therapeutic effect. Dual-specific or “Two-in-One” antibodies can bind to two different antigens separately with the same antigen-binding site as opposed to bispecifics, which simultaneously bind to two different antigens through separate antigen-binding units. These nonstandard dual-specific antibodies were recently shown to be promising for new antibody-based therapeutics. Here, we physicochemically and structurally analyzed six different antibodies of which two are monospecific and four are dual-specific antibodies derived from monospecific templates to gain insight about dual-specificity determinants. These dual-specific antibodies can target both human epidermal growth factor receptor 2 and vascular endothelial growth factor at different binding affinities. We showed that a particular region of clustered Vernier zone residues might play key roles in gaining dual specificity. While there are minimal intramolecular interactions between a certain Vernier zone region, namely LV4 and LCDR1 of monospecific template, there is a significant structural change and consequently close contact formation between LV4-LCDR1 loops of derived dual-specific antibodies. Although Vernier zone residues were previously shown to be important for humanization applications, they are mostly underestimated in the literature. Here, we also aim to resurrect Vernier zone residues for antibody engineering efforts.     

At least two loci encode polymorphic class I MHC antigens in the horse

Summary. Six monoclonal antibodies and ten alloantisera were used to precipitate cell surface molecules of approximately 44kDa (class I MHC antigens) from radiolabelled equine peripheral blood lymphocytes. All ten antisera were raised against antigens of a single donor horse (horse 0834, ELA-A2,-A2). Four methods of producing antisera were compared: one or two pregnancies, skin allografting, and skin grafting followed by pregnancy. Immunization by pregnancy appeared to produce antibodies against class I products only, while skin grafting raised antibodies to class II antigens as well. Nine of the antisera were raised across an entire MHC haplotype barrier, while one recipient carried the ELA-A2 antigen of the donor. The pregnancy antiserum raised across this barrier probably identifies a second polymorphic class I locus in the horse. Sequential immunoprecipitation using this antiserum in the first stage and an anti-MHC haplotype antiserum or monoclonal antibody reagent in the second stage supported this hypothesis. Gene products of this second ELA class I locus are immunogenic in pregnancy.     

One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.     

Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid,choline acetyltransferase or serotonin

Summary In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gammaamino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of primary antisera, the sections are incubated in a cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H₂O₂ (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H₂O₂ (brown reaction product in the target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.     

Comparison of different double immunostaining protocols for paraffin embedded liver tissue.

Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue-specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non-specific background staining in pathological liver specimens. We compared peroxidase-anti-peroxidase, alkaline phosphatase-anti-alkaline phosphatase (PAP/APAP), labelled-avidin-biotin (LAB/LAB) and digoxigenin-anti-digoxigenin (dig-a-dig/PAP) techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP-technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig-a-dig/PAP protocol. In contrast to the dig-a-dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue.     

Limitation of microwave treatment for double immunolabelling with antibodies of the same species and isotype

Microwave treatment (MW) involves completely blocking contaminating staining in the double-labelling technique, using primary monoclonal antibodies from the same species and the same isotype as well as the same secondary antibody (ab). However, we noticed some limitations when locating proliferating cell types in cryostat and paraffin sections using the advantages presented by MW. Control experiments have shown that MW does not diminish contaminating staining when cytoplasmic (desmin, ASM-1) or nuclear (Ki-67) antigens have been labelled with antibodies in the first round of immunolabelling. In contrast to the cell surface antigen, CD18, where the primary ab had to be crosslinked by a secondary ab to obtain contaminating staining, this was observed for the detection of cytoplasmic or nuclear antigens only labelled with a primary ab. In conclusion, for double immunolabelling with abs from the same species and the same isotype, MW is not able to completely abolish contaminating staining.     

A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride

Very few double-antigen staining methods are available that are applicable to both light and electron microscopy. The objective of this study was to develop for localization of two neural antigens simultaneously a procedure which would be sensitive, simple to perform, offer permanent reaction products, and permit correlated light and ultrastructural analysis. The method employs sequential immunoperoxidase staining without antibody elution, in which the first sequence of antibodies is visualized with 3,3'-diaminobenzidine (DAB) and the second with benzidine dihydrochloride (BDHC). The DAB reaction product (brown and diffuse) was easily distinguishable from the BDHC deposit (blue, granular, and more electron-dense) by both light and electron microscopy. The procedure was used to simultaneously localize choline acetyltransferase-and either substance P or tyrosine hydroxylase in rat brain at both light and ultrastructural levels. Control experiments demonstrated the absence of both color mixing and antibody crossreactions, even when both primary antibodies were from the same species. This study demonstrates the usefulness of BDHC as a chromogen for immunoperoxidase staining either alone or in combination with DAB, and describes a double method which should have wide applicability for detailed studies of most pairs of antigens at both light and ultrastructural levels.