Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.     

Finding the best antibody dilution by repeated immunostaining of the same tissue section

One can find the optimal antibody dilution for immunostaining by repeated staining on the same tissue section by using a less dilute antibody for each attempt. Using secondary antibody and horseradish peroxidase conjugated to a dextran polymer, a section stained repeatedly with several dilutions of antibody appears as good as a section stained with only the last dilution.     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

The labeled antigen method of immunoenzymatic staining.

A two stage immunohistological technique (the "labeled antigen" procedure) has been assessed for the detection of a variety of human and animal cytoplasmic constituents in tissue sections. In this method specific antiserum is followed by antigen complexed to horseradish peroxidase or to alkaline phosphatase. The primary antibody acts bivalently, linking the labeled antigen to antigen in the tissue section. The major advantage of this technique is that nonantigen specific antibody in the primary antiserum cannot cause nonspecific staining since it has no affinity for the antigen:enzyme complex. Consequently the specificity of the reaction is assured, background staining is minimized and the total staining time (from wax section to mounted slide) can be reduced to as little as 30 min. Further advantages include the possibility of labeling Ig allotypes and the high efficiency of enzyme utilization. Covalent human IgG:horseradish peroxidase complexes can also be used in a triple sandwich in conjunction with human anti-viral or autoimmune antibodies.     

Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.     

The effects of magnesium and calcium ions on phosphatase activities at alkaline pH in the molar region of newborn mice

Summary The hydrolysis of ATP, AMP and glycerophosphate (GP) at alkaline pH in mineralizing bone and teeth of young mice has been studied histochemically. The substrates were visibly hydrolyzed to the same degree in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at Ca²⁺ concentrations ranging from 10 mM to 600 mM. In the ameloblasts, however, only ATP was hydrolyzed. The ATPase activities gradually decreased at increasing Mg²⁺/Ca²⁺ ratios. The AMPase and GPase activities, on the other hand, were visibly unaffected. Marked cellular staining, including the nuclei was seen with AMP and GP as substrates when only Mg²⁺ ions were added. No ATPase activity at all could be recorded in media containing Mg²⁺ but no Ca²⁺ ions. The different phosphatase activities in cells involved in hard tissue formation were identically affected by preincubations with solutions containing various concentrations of Ca²⁺ or Mg²⁺ ions. The ATPase activity in striated muscle fibres and blood vessel walls, however, was affected differently by the same procedure.The results indicate that the phosphatase activities recorded in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at alkaline pH belong to one single enzyme. The results also imply that CaATP is the preferred substrate in the enzymatic hydrolysis of ATP in hard-tissue-forming cells.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Summary In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using³⁵S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using³⁵S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Direct immunoenzyme double staining applicable for monoclonal antibodies

Summary A novel method for immunoenzymatic double staining was developed, using primary antibodies directly labeled with either horseradish peroxidase or alkaline phosphatase. The enzyme-antibody conjugates were applied simultaneously on sections of human tonsil. Intracytoplasmic antigens like immunoglobulins and light chains could easily be detected simultaneously in the same tissue section. With antibodies against cell surface antigens like IgM and T cell antigens areas containing B and T cells could be clearly distinghuished. This method opens the possibility to perform double staining with two monoclonal antibodies.     

Indirect immunofluorescence modified to display two antigens with one light filter

Summary We present a modification of double indirect immunofluorescence in which we used four antibodies raised in three species to visualize two different antigens. The procedure, which relies on dual recognition of a secondary antibody, requires that one primary antibody and one of the secondary antibodies be raised in the same species. As the two secondary antibodies are conjugated to two different fluorochromes, both of the antigens studied are visualized with one light filter while only one antigen is displayed with another filter. This, in turn, allows more efficient comparison of the distribution of the two antigens in a single field or photograph than is possible by comparing two fields or photographs by conventional double staining. The method is especially useful for determining possible co-localization of two cellular structures. We illustrate the method in adrenal cells in which mitochondria and intermediate filaments are seen to be co-localized.     

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell ¹.Open in a separate windowClick here to view.^{(64M, flv)}     

Light and electron microscopical combined staining by immunohistochemical and enzyme histochemical methods using rat prolactin-secreting pituitary tumours

Summary Combined immunohistochemical staining (IHCS) and enzyme histochemical staining (EHCS) methods for light microscopy (LM) and electron microscopy (EM) are reported, using oestrogeninduced rat pituitary tumours. For LM, combined staining for alkaline phosphatase and acid phosphatase by EHCS, using the azo dye method, and for prolactin and ACTH by IHCS, using the enzyme-labelled antibody method, gave the best results on 1 m glycol methacrylate sections. For EM, combined staining by EHCS on 30 m tissue sections followed by IHCS for prolactin on ultrathin Epon sections (enzyme-labelled antibody method) provided acceptable results. By these combined staining methods, the neoplastic prolactin cells were shown to have close affinity to rich alkaline phosphatase-positive capillaries and to possess an alkaline phosphatase-positive cell membrane. Furthermore, they revealed acid phosphatase-positive lysosomal and secretory granules. These combined staining methods may be valuable in studies on the actual functional status of cells.     

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:     

Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

Production of an antibody to arterial fatty acid-binding protein (FABP) using a synthetic peptide as the antigen.

1. A procedure is described for the preparation of an antibody to arterial FABP using a synthetic peptide as an antigen. In order to locate a highly conserved region located on the outer surface of FABP, computer analysis of primary and secondary structures of several proteins from the FABP family was undertaken and a 24 amino acid sequence beginning at the fifth position from the N-terminus of rat heart FABP was chosen. 2. The synthetic peptide consisted of eight replications of the 24 amino acid sequence individually attached to the alpha and epsilon amino groups of each terminal lysine on an octalysine branched peptide. 3. Antibody to the synthetic antigen was raised in New Zealand rabbits. Western analysis was conducted and detection was accomplished by using goat-anti-rabbit second antibody conjugated to alkaline phosphatase. 4. The antibody produced from the previously described peptide, recognized purified rat heart FABP and demonstrated a high positive correlation (r = 0.96) when known concentrations of purified hFABP were plotted against densitometric measurement of the bands. 5. Additionally, the antibody recognized FABP from the 104,000 g supernates of rat atrial and arterial tissue fractionated by a Sephadex G-75 column. 6. Therefore, the antibody produced from this particular protocol employing a synthetic peptide can be utilized qualitatively and quantitatively in the analysis of the heart and arterial FABP content.     

Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18⁺/PSA^– HT29 colon carcinoma cells. CK18⁺ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy. In conclusion, the approach presented appears to be a reliable method to phenotype individual prostatic carcinoma cells.     

Genetic studies of human apolipoproteins. V. A novel rapid procedure to screen apolipoprotein E polymorphism

A simple and new method has been developed to detect apolipoprotein E polymorphism directly from plasma or serum without prior ultracentrifugation and delipidation. The method combines the use of dialyzed plasma or serum samples with or without neuraminidase treatment followed by monodimensional isoelectric focusing in simple or 3 M urea gels at a constant low power and progressively increasing voltage over a 3-hr period, and finally protein blotting to a nitrocellulose membrane. Apolipoprotein E phenotypes are identified immunologically using a double antibody reaction, the primary antibody being a monospecific, polyclonal goat anti-apolipoprotein E, and the secondary antibody being a rabbit anti-goat IgG conjugated with alkaline phosphatase. The method was employed to screen apolipoprotein E polymorphism in two white populations in the United States. The frequency values are comparable to those reported previously by other investigators using conventional detection methods. The procedure is simple, accurate, suitable for large scale epidemiologic, clinical, and genetic studies.     

Covalent binding sites of victorin in oat leaf tissues detected by anti-victorin polyclonal antibodies

Polyclonal antibodies against victorin, the host-specific toxin produced by Cochliobolus victoriae, were raised in rabbits immunized with a victorin-bovine serum albumin conjugate. The antibodies were purified from serum by protein A column chromatography and characterized by indirect and direct enzymelinked immunosorbent assays (ELISA). The concentration of victorin that inhibited anti-victorin antibody binding by 50% was 10 nanograms per milliliter in an indirect ELISA. The lowest concentration of victorin detectable was 10 picograms per milliliter. In a direct ELISA, 25 nanograms per milliliter of victorin inhibited binding of victorin-horseradish peroxidase conjugate by 50%. In vivo and in vitro covalent binding of victorin to proteins in susceptible and resistant oat (Avena sativa) tissue was examined by western blotting assays using anti-victorin antibody and a second antibody conjugated with ¹²⁵I or alkaline phosphatase. In vivo binding of victorin to proteins of 100 and 45 kilodaltons was observed in both susceptible and resistant cultivars of oats. Victorin also bound in vitro to proteins of 100, 65, and 45 kilodaltons in both susceptible and resistant oats. The data indicate that victorin binds covalently to the same sites in susceptible and resistant genotypes of oats.     

Color-Contrast Staining of Two Different Lymphocyte Subpopulations: A Two-Color Modification of Alkaline Phosphatase Monoclonal Anti-Alkaline Phosphatase Complex Technique

A dual staining method for different human lymphocyte subpopulations with nonoverlapping antigen distribution patterns is described. Cytocentrifuge slide preparations of peripheral blood nonadherant mononuclear cells (NAMNC), bone marrow aspirate or buffy coat smears were fixed in acetone and incubated with a primary mouse monoclonal antibody (MAb) against a lymphocyte antigen (CD8, Ig-light-chain, CD19, CD4) followed by rabbit anti-mouse immunoglobulin (Ig) and the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) complex. After repeating the “bridge” antibody and the APAAP, a red product was developed with fast red TR-naphthol AS-BI phosphate. Following this one-color stain the process was repeated using a different primary mouse MAb against another lymphocyte antigen (CD4, Ig-light chain, CDS, MHCU DR, CD5) and fast blue BB-naphthol AS-MX phosphate at the last step to yield a blue product. Control slides stained by the standard one-color APAAP method with die relevant primary MAb showed that there was no nonspecific labelling and the percent of positive cells in a given test was almost identical. To achieve an intense blue in the second stain for some antigens, e.g., CD4, either the MAb concentration had to be increased or two different MAbs recognizing differing epitopes of the same antigen, e.g., T1 and UCHT2 for CD5, were applied. Any change of red to purple at the site of the first stain after 15 min exposure to the blue-yielding AP substrate is due to residual AP activity of the first stain rather than to crossbinding of immunoreagents. This rechnique allows sensitive two-color staining without being limited to the few antigens against which heteroantibodies are available. High nonspecific background staining due to endogenous enzyme activity, as inherent in peroxidase staining procedures, is overcome in this dual APAAP technique.     

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.     

Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi- or trans-illuminators combined with photographic or charge-coupled device cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.