Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry

Summary Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3-diaminobenzidine (DAB),p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co²⁺ and Ni²⁺ ions. The results indicate that DAB-imidazole and DAB-Co²⁺ and Ni²⁺ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.     

Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies

Although very sensitive chromogens have been adapted for localization of horseradish peroxidase in anterograde and retrograde tracing studies, they have not been successfully applied in immunocytochemical studies. This report describes a protocol which uses benzidine dihydrochloride (BDHC) as the chromogen for light (LM) and electron microscopic (EM) immunocytochemical studies. The protocol is comparable to that used for tetramethylbenzidine, except that the pH of the reaction is above 6.0. At the LM level, the BDHC reaction product is bluish-green and crystalline. Both the color and form of the product are readily distinguished from the reddish-brown DAB reaction product. LM double-labeling studies are therefore feasible. The use of BDHC also increases significantly the sensitivity of the immunoreaction. Higher fixative concentrations can be used, less detergent is necessary, and higher primary antibody dilutions are possible. By osmicating at 45 degrees C in an s-collidine buffer it is possible to preserve the soluble BDHC reaction product for EM analysis. Immunoreactive cells are particularly well labeled with this new protocol. The BDHC crystals are easily detected at the EM level and can be distinguished from flocculent DAB reaction product. This feature makes EM double-labeling studies possible.     

Distribution of neurofilament subunits in neurons and neuronal processes: immunohistochemical studies of bovine cerebellum with subunit-specific monoclonal antibodies

The distribution of individual neurofilament (NF) subunits in bovine cerebellar neurons was examined using monoclonal antibodies (MAs) raised against bovine NF. MAs with immunochemically defined specificities for one or more NF subunits were used. Seven were specific for the Mr 68,000 NF subunit, five were specific for the Mr 150,000 NF subunit, nine were specific for the Mr 200,000 NF subunit, and 30 recognized both high molecular weight subunits. Fresh bovine cerebellum was fixed and processed by five different protocols and subjected to four different immunohistochemical procedures. MAs from each group stained neuronal perikarya and processes. NF immunoreactivity in Purkinje cells was evaluated in detail. Adjacent Purkinje cell bodies and dendrites exhibited variable NF immunoreactivity to the same MA, ranging from intensely positive to completely negative. Similar variability in axonal staining was not observed. Application of the same MA to tissue subjected to different fixation and/or immunohistochemical protocols also resulted in variability in NF subunit immunoreactivity. We conclude that MAs recognize each of the three NF subunits in neuronal perikarya, axons, and dendrites. Variability in NF subunit immunoreactivity appears to reflect both NF microheterogeneity and fixation-dependent modifications of NF subunits.     

A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.     

A simple enhancement method for the silver-gold-intensified diaminobenzidine reaction in the light microscopic immunoperoxidase technique.

We describe a simple and sensitive method for enhancement of the silver-gold-intensified 3,3'-diaminobenzidine (DAB) reaction demonstrating peroxidase activity. After completing silver-gold intensification of the preparations immunostained by the avidin-biotin-peroxidase method with DAB as the chromogen, the preparations were immersed in a solution containing uranyl nitrate. This new method appeared to increase the sensitivity by at least one order of magnitude as compared with silver-gold intensification alone.     

A dual-immunocytochemical method to localize c-fos protein in specific neurons based on their content of neuropeptides and connectivity

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 m in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP). Secondly, to identify activated neurons in female Syrian hamsters at the time of the proestrous luteinizing hormone surge, c-fos protein staining with DAB was carried out in combination with BDHC staining for gonadotrophin-releasing hormone (GnRH). In both these studies, cells which co-localized the peptide and c-fos protein in the nucleus could be identified unequivocally. Thirdly, to analyse projections of c-fos-immunoreactive neurons, the retrograde tracer, cholera toxin subunit B (ChB) was pressure-injected into the piriform cortex of rats, which were thereafter fully kindled in the contralateral amygdala. The tract tracer was stained with BDHC as the chromogen. Due to the advantages of the dual-labelling methodology, the combination of retrograde tracing and c-fos protein histochemistry provides an excellent method for identifying projecting and activated neurons in the same section.     

Notes on the combined use of V-VIP and DAB peroxidase substrates for the detection of colocalising antigens

 The purpose of the present report was to investigate to what extent the new peroxidase substrate Vector VIP (V-VIP) can be used in combination with DAB chromogen for the unequivocal and permanent detection of colocalising antigens within a single neurone, according to a two-colour paradigm. With this aim, re-trograde tract-tracing with cholera toxin B subunit (CTB) or fluoro-gold (FG) was performed to disclose individual, identified subpopulations of neurones in the primate substantia nigra projecting to the caudate nucleus or to the putamen, respectively. Each tracer was detected by means of a PAP procedure and finally stained brown using DAB as a chromogen. Subsequently, both series of sections were processed for the immunocytochemical detection of tyrosine hydroxylase (TH). TH-immunoreactive neurones were stained purple with the peroxidase substrate V-VIP. As a result of the present procedure, several cell bodies of projection neurones, stained brown, can easily be identified within the primate substantia nigra. Some of these neurones additionally displayed purple TH immunoreaction product located in the neuronal dendrites. By contrast, CTB- or FG-unlabelled neurones only show the typical purple precipitate that belongs to V-VIP substrate, both in the cell body as well as in the dendrites. Accepted: 20 January 1999     

Neurofilament phosphorylation in development. A sign of axonal maturation?

Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation.     

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.     

New Silver-Gold Intensification Method of Diaminobenzidine for Double-Labeling Immunoelectron Microscopy

The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3′-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level.     

Presence of an alpha-melanocyte-stimulating hormone-like epitope in the 150,000 dalton neurofilament subunit from diverse regions of the central nervous system: an immunohistochemical and immunoblot study in guinea pig

Monoclonal antibodies specific for the two higher molecular weight neurofilament (NF) subunits (NF200 and NF150), and antiserum to alpha-melanocyte-stimulating hormone (alpha-MSH) were used to probe the distribution of an alpha-MSH-like epitope in NF proteins of the guinea pig central nervous system using immunoblot and immunohistochemical methods. The anti-alpha-MSH antiserum recognized the same protein band as an anti-NF150 monoclonal antibody in immunoblots of proteins extracted from guinea pig cerebellum, spinal cord, retina, optic nerve, and neurohypophysis; it also stained axons and dendrites in sections of cerebellum, retina, and optic nerve. Although all cells of the pars intermedia and some in the pars distalis exhibited immunoreactivity with this antiserum, it did not stain axons in the neurohypophysis. Our immunoblot data demonstrate an alpha-MSH-like epitope in NF150 extracted from each of the regions studied. The lack of in situ recognition of this alpha-MSH-like epitope in neurophypophyseal axons, using the same immunohistochemical methods that demonstrate this epitope in axons of the cerebellum, retina, and optic nerve, suggests that NF150 is immunochemically heterogeneous in different regions of the guinea pig central nervous system.     

Amplification of the in situ hybridization signal by silver postintensification: the biotin-dUTP-streptavidin-peroxidase diaminobenzidine-silver-gold detection system

Summary Frozen and vibratome sections from the adrenal gland of the rat were hybridized in situ using a biotinylated oligonucleotide probe specific for tyrosine hydroxylase (TH) messenger ribonucleic acid (mRNA). Hybridization was detected using the streptavidin-peroxidase-diaminobenzidine (DAB) system in combination with silver-gold postintensification. The signal appeared as a black coloration and was localized to the cytoplasm of catecholamine-synthesizing chromaffin cells in the adrenal medulla. This coloration was due to the deposition of the silver-gold intensified DAB chromogen onto the probe hybridized to mRNA in carrier organelles. Compared with the conventional peroxidase-DAB labelling, the silver-gold amplified version was more sensitive in detecting TH mRNA. Using this modification, we were able to adapt the procedure to electron microscopy, thereby further localizing the hybridized signal to ribosomes. Because this hybridization detection system produces grains, not just color, this method has the potential for measurement of changes in mRNA levels at the ultrastructural level.     

Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures

We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.     

Binding of copper(II) to carnosine: Raman and IR spectroscopic study

A comparative Raman and FTIR study of carnosine, a dipeptide present in several mammalian tissues, and its complexes with copper(II) at different pH values was carried out. The neutral imidazole ring gives rise to some bands that appear at different wavenumbers, depending on whether the imidazole ring is in the tautomeric form II or I. At pH 7 and 9 the molecule exists in equilibrium between the two tautomeric forms; tautomer I is predominant. Metal coordination is a factor that affects the tautomeric equilibrium, and the copper(II) coordination site can be monitored by using some Raman marker bands such as the vC(4)=C(5) band. On the basis of the vibrational results, conclusions can be drawn on the functional groups involved in the Cu(II) chelation and on the species existing in the Cu(II)-carnosine system. At neutral and basic pH the most relevant species formed when the Cu(II)/carnosine molar ratio is not very different from unity is a dimer, [Cu(2)L(2)H(-2)](0). In this complex the ligand coordinates the metal via the N (amino), O (carboxylate), and N (amide) donor atoms while the N(tau) nitrogen atoms of the imidazole rings (tautomer II) bridge the copper(II) ions. At a slightly acidic pH the two monomeric complexes [CuLH](2+) and [CuL](+) were present. In the former the imidazole ring takes part in the Cu(II) coordination in the tautomeric I form whereas in the latter it is protonated and not bound to Cu(II).     

Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system

A number of methods for demonstration of peroxidase activity have been tested on immunocytochemical nitrocellulose models. By applying a well-characterized primary antibody and Sternberger's peroxidase-antiperoxidase technique, the sensitivity of various protocols has been evaluated. Best results were obtained with diaminobenzidine as chromogen, especially in conjunction with heavy metal salts, either added directly to the medium or used as "toners" of the end-product. Use of silver intensification of the diaminobenzidine/metal end-product increased sensitivity further.     

Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study.

The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types.     

Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog.

Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes. Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer. Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium. Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions. The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding. The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody. In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro. Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants. The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins.     

Diaminobenzidine Induces Fluorescence in Nervous Tissue and Provides Intrinsic Counterstaining of Sections Prepared for Peroxidase Histochemistry

3,3'-Diaminobenzidine (DAB) is widely used as a chromogen for visualization of horseradish peroxidase activity in neuroanatomical tracing experiments and in immunohistochemistry. The product of the enzymatically catalyzed oxidation of DAB by hydrogen peroxide is brown and nonfluorescent. In frozen sections of formaldehyde fixed rat and mouse brain that had been exposed to DAB either alone or with hydrogen peroxide, we observed strong greenish fluorescence in myelinated nerve fibers and in the somata of some neurons. This fluorescence was not associated with brown coloration and was not due to endogenous peroxidase activity. Extractions, blocking reactions, and other histochemical tests indicate that the fluorescence resulted from the combination of DAB with aldehyde groups that were formed by oxidation of unsaturated linkages in lipids. DAB induced fluorescence provides a simple and useful demonstration of background anatomy in sections that also contain specifically localized deposits of peroxidase activity.     

Purification and characterization of a novel (R)-imine reductase from Streptomyces sp. GF3587

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5-8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10-11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.     

Coupled oxidation of NADPH with thiols at neutral pH.

NADPH and NADH are rapidly oxidized in neutral imidazole chloride buffer at 30 °C in the presence of mercaptoethanol or dithiothreitol. The product of the NADPH reaction has been determined to be enzymically active NADP⁺. Oxidation of the pyridine nucleotides is coupled to the autooxidation of the thiol and is inhibited by ethylenediamine tetraacetic acid, stimulated by metal ions (FeSO₄), and requires oxygen. The rapid oxidation of thiols and NADPH at neutral pH was found to occur only in imidazole and, to a lesser extent, in histidine buffer. Under the conditions employed, 300 μm dithiothreitol and 30 μm NADPH are oxidized in 30 min. Both NADPH and thiol oxidations are inhibited by catalase, whereas superoxide dismutase only inhibits the oxidation of NADPH. NADPH oxidation is also inhibited by the hydroxyl radical scavengers formate, mannitol, or benzoate. A reaction mechanism is proposed in which imidazole promotes the metal-catalyzed oxidation of thiols at neutral pH. The superoxide radical generated either by the thiol oxidation or directly oxidizes NADPH or forms hydrogen peroxide and hydroxyl radicals which can oxidize NADPH. Hydrogen peroxide is also involved in the autooxidation of the thiol.