Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase. Applications in immunoassays

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2.Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG1 and IgG2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

Summary The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP₂·Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG₁ and IgG_2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin. This study demonstrates some of the potential advantages of using an epitope specific monoclonal bi-specific developing reagent like McC10 in an immunobased technique like ICC. Its potential use in a variety of other immunobased procedures is discussed.     

A comparison of the ability of serum and monoclonal antibodies to gastric inhibitory polypeptide to detect immunoreactive cells in the gastroenteropancreatic system of mammals and reptiles

Summary A monoclonal antibody raised to gastric inhibitory polypeptide (GIP) has been compared with conventional rabbit and guinea-pig antisera to GIP. Four staining methods were tested and of these the peroxidase antiperoxidase (PAP) method proved to give the best results with both the mouse and rabbit antibodies. The monoclonal antibody, when used to stain pancreatic tissue, gave negative results whereas a distinct population of gut endocrine cells was readily demonstrable, suggesting that GIP is not a constituent of the mammalian pancreas. The monoclonal antibody was found to be the most sensitive for immunocytochemistry achieving the titre of 1:10⁶ in rat gut. A C-terminal specific antibody, with a high affinity and avidity to GIP, it was clearly the preferred antibody for immunocytochemical studies.     

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specific antibodies.     

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Summary Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specifie antibodies.     

Monoclonal antibodies against amyloid fibril protein AA. Production, specificity, and use for immunohistochemical localization and classification of AA-type amyloidosis

Monoclonal antibodies against amyloid fibril protein AA were produced by cell fusion of murine P3 X 63-Ag8.653 myeloma cells with spleen cells of immunized Balb/c mice. To increase immunogenicity, protein AA was coupled to horseradish peroxidase (HRP) or human high molecular weight kininogen (HMWK). Using micro-ELISA (enzyme-linked immunosorbent essay) seven hybridoma cell lines secreting antibodies that specifically bind to protein AA have been selected and cloned. When applied to formalin-fixed paraffin sections of a variety of different amyloid types using immunoperoxidase methods, five monoclonal antibodies bound specifically and strongly to amyloid only of the AA type. Since a series of different AA-amyloids could be stained, these reagents may be used to routinely diagnose AA-amyloidosis in tissue sections. A monoclonal antibody against HRP has also been produced that has been utilized to develop a monoclonal peroxidase-antiperoxidases (PAP) complex. When three immunoperoxidase methods were compared, the sensitivity of a conventional rat PAP was comparable to the monoclonal PAP complex, but the latter was easier to handle. Both methods were more sensitive than the indirect immunoperoxidase technique.     

Immunocytochemistry with monoclonal anti-immunoglobulin as a developing reagent: application to immunoperoxidase staining and radioimmunocytochemistry

The development of two monoclonal antibodies for use as second antibodies in immunocytochemistry is described. The antibodies are produced by mouse X mouse hybrid myelomas, and are both of the IgG type. The two antibodies, RB23 and ND13, were used to detect neurophysin by three-step peroxidase-antiperoxidase (PAP) immunostaining, and were "internally labeled" with 3H-lysine for the radioimmunocytochemical localization of neurophysin, substance P, and tyrosine hydroxylase using rabbit first antibodies. The binding sites of RB23 and ND13 on the rabbit IgG antibodies were determined by solid-phase radioimmunoassay, using allotype-specific rabbit serum to compete with RB23 and ND13. It was found that both RB23 and ND13 are directed against the B4 kappa-light-chain allotype. The immunocytochemical localization of adrenocorticotropic hormone and somatostatin with rabbit primary antibodies was not achieved with RB23 or ND13, and it is proposed that these antibodies are not of the B4 allotype. The findings demonstrate that monoclonal second antibodies can be useful general reagents for conventional immunocytochemistry as well as for radioimmunocytochemistry. Furthermore, allotype-specific monoclonal second antibodies may prove useful in the simultaneous immunohistochemical localization of more than one antigen in a given tissue section.     

Preparation of peroxidase: antiperoxidase (PAP) complexes for immunohistological labeling of monoclonal antibodies

The production of mouse peroxidase:antiperoxidase (PAP) complexes suitable for immunohistological use in conjunction with monoclonal antibodies is described. Three approaches were explored: 1) production of conventional polyclonal PAP complexes; 2) conversion of rabbit PAP to "pseudo-mouse PAP" by incubation with monoclonal mouse anti-rabbit immunoglobulin; 3) formation of PAP complexes from monoclonal mouse antiperoxidase. PAP complexes prepared by the latter technique gave the best immunohistological labeling reactions, being stable on storage and compatible with a wide range of human monoclonal antibodies. Gel filtration revealed that monoclonal PAP is of lower molecular weight than conventional PAP complexes (fulfilling theoretical predictions based on the monospecificity of monoclonal antibodies).     

Radioimmunocytochemistry using a tritiated goat anti-rabbit second antibody

Affinity-purified goat anti-rabbit immunoglobulin G (GAR) was conjugated with (3H)-propionyl succinimidate and used to localize substance P (SP), enkephalin (ENK), and serotonin immunoreactive sites in the spinal dorsal horn and medulla of the rat and cat. Autoradiographic localization was demonstrated on paraffin, frozen, Vibratome, and 2 micron plastic sections. The latter were obtained from radiolabeled Vibratome sections that were embedded in epoxy resin. The distribution of SP, ENK, and serotonin demonstrated by radioimmunocytochemistry was comparable to that observed on semiadjacent sections using peroxidase-antiperoxidase (PAP) immunocytochemistry. The autoradiograms, however, were generated using primary antibody concentrations up to five times more dilute than concentrations used for the PAP procedure. Indirect radioimmunocytochemistry using a (3H) anti-immunoglobulin G second antibody can be used to localize a variety of monoclonal and polyclonal antisera. It is quantifiable at the light microscopic level and can be potentially used with peroxidase histochemistry to double label immunoreactive structures at the ultrastructural level.     

Development of a bi-specific monoclonal antibody for simultaneous detection of rabbit IgG and horseradish peroxidase: use as a general reagent in immunocytochemistry and enzyme-linked immunosorbent assay

We describe the development of bi-specific monoclonal antibodies (MAb) capable of simultaneous recognition of rabbit immunoglobulin G (IgG) and horseradish peroxidase (HRP) for use in a variety of immunobased techniques. This bi-specific antibody, named McC8, was produced by fusion of the aminopterin-sensitive mouse hybridoma MAP.Ag.1, which secretes MAb against HRP, and splenocytes from a mouse previously immunized with whole rabbit IgG. The resultant hybrid-hybridoma co-dominantly expresses and secretes the immunoglobulin chains, i.e., IgG1 and IgG2b, of its respective parents, as determined by radial immunodiffusion. The binding sites on rabbit IgG for McC8 were determined on Western blots and in competition solid-phase enzymatic immunoassays with the use of allotype-specific rabbit sera. Both these techniques demonstrated that McC8 recognizes the light chain of the rabbit IgG molecule with preferential binding to the B4 kappa light-chain allotype. McC8 was successfully used in two-step immunocytochemistry for localization of calcitonin gene-related peptide (CGRP) in fibers of the superficial layers of the spinal trigeminal nucleus of the rat, as well as for localization of glial fibrillary acidic protein (GFAP)-immunoreactive sites in primary rat septal cell cultures, thus demonstrating its potential as a general developing reagent in conventional immunocytochemistry. McC8 compared favorably with peroxidase-antiperoxidase immunocytochemistry with respect to sensitivity. However, the bi-specific developing reagent proved superior to the conventional peroxidase-antiperoxidase procedure when both were employed in a similar fashion in tissues prone to display high background staining. Finally, McC8 was also employed as a developing reagent in a competitive ELISA designed for quantitation of CGRP with the use of a rabbit anti-CGRP primary antibody. The sensitivity of this quantitative ELISA (190 pg or 50 fmol CGRP per well) renders this bi-specific antibody suitable for use in quantitative immunoassays for detection of relevant peptides in biological systems.     

Enhanced immunocytochemical staining sensitivity in formalin-fixed and paraffin-embedded material by simultaneous use of PAP and ABC

The simultaneous use of two immunoperoxidase (IP) methods; e.g. the PAP (Peroxidase-antiperoxidase) and ABC (Avidin-Biotin) to localize a single antigen enhances the sensitivity of polyclonal antibodies (FVIII/RAg and laminin) or monoclonal antibodies (MABs) (against human B-, T-cells and macrophages) in routinely formalin fixed and in paraffin embedded material. The increased sensitivity was not accompanied by loss of specificity. With antibodies against Factor VIII related antigen (FVIII/RAg) and laminin the increased staining intensity of blood vessels were demonstrated in experimental rat transplantation tumors. The similar enhancement of immunocytochemical reactions was observed with biopsies of human brain tumors, where the monoclonal antibodies against white blood cells were used. The staining results were of comparable intensity as in snapfrozen tissue or after special embedding procedure for immunocytochemical purposes acc. to Bolton and Mesnard formol sucrose gum, sucrose paraffin; FSGSP).     

Combination of the peroxidase anti-peroxidase (PAP)- and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step--Incubation of the tissue sections with the monoclonal primary antibodies; Second step--biotinylated anti-rat or anti-mouse IgG; Third step--monoclonal PAP complex; Fourth step--ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step--the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4- and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.     

Anti-angiotensin II anti-idiotypic antibodies bind to angiotensin II receptor

An anti-idiotypic serum from a rabbit immunized with one anti-angiotensin II (AII) monoclonal antibody (A25) was shown to identify a cross-reactive idiotope (CRI) shared by six anti-AII monoclonal antibodies, in addition to a binding site-associated private idiotope. This anti-idiotypic reagent bound to rat liver membranes bearing AII receptors; binding was abolished after pretreatment of the membranes with AII. In immunoblotting experiments with rat liver membranes, as well as with rat pituitary homogenates, a 63,000 +/- 2,000 dalton protein was revealed that co-migrated with the AII receptor. After purification by affinity chromatography on an immobilized CRI+-antibody (A41), anti-CRI antibodies could immunoprecipitate the hormone binding activity from detergent-treated rat liver membranes and still recognize the 63,000 dalton protein. In contrast, anti-idiotypic antibodies specific for the private idiotope failed to interact with the AII receptor. Similar results were obtained with a second anti-idiotypic serum produced by immunization with another CRI+ anti-AII monoclonal antibody (A22). The sharing of the CRI determinant between the AII receptor and anti-AII antibodies might account for the reactivity of anti-idiotypic antibodies towards the AII receptor.     

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.     

A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues

We compared the streptavidin-peroxidase conjugate (SP) method of immunoperoxidase histochemistry to the unlabeled antibody (PAP) and avidin-biotin-peroxidase complex (ABC) techniques in human colorectal carcinoma tissues stained with a monoclonal antibody for expression of carcinoembryonic antigen. Compared to the ABC and PAP method, the SP method produced stronger staining intensity and very low background staining. This was true when other antibody isotypes, other antibody species, other organs, and another tumor-associated antigen were used. Moreover, the SP procedure time could be reduced to one third that of the ABC or PAP methods without compromising accuracy, and the SP reagent is stable for several months. The chemical nature of the streptavidin molecule accounts, in large part, for the advantages of the SP method.     

Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin.

Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues. (J Histochem Cytochem 47:1237-1245, 1999)     

Improved immunocytochemical staining through the use of Fab fragments of primary antibody, Fab-specific second antibody, and Fab-horseradish peroxidase

A modification of the unlabeled antibody method of immunocytochemistry is described here that offers increased immunoreagent penetration and greatly reduced background staining. The method involves the following alterations to the conventional technique; the use of Fab fragments of primary antibody, rather than whole immunoglobulin G (IgG) or serum; the use of a second, or link, anti-rabbit IgG serum that is Fab fragment-specific, rather than directed against the whole rabbit IgG molecule; the use of the Fab--horseradish peroxidase complex described by JR Slemmon, PM Salvaterra, and K Saito (J Histochem Cytochem 28:10, 1980), rather than peroxidase--antiperoxidase (PAP). Steps 2 and 3 alone brought about a significant reduction in background staining, but did not increase the depth of immunostaining, as compared to the PAP technique. When all three steps were combined, however, background staining was further reduced, and there was a five- to tenfold increase in the depth of immunostaining. These readily made changes should be useful in preembedding immunocytochemistry whenever enhanced reagent penetration is required.     

Monoclonal antibody to human lysosomal alpha-glucosidase in immunocytochemistry: unexpected reactivity with cytoskeletal structures

Summary A well-characterized monoclonal antibody against human lysosomal -glucosidase has been used for the immunohistochemical localization of the enzyme in cultured human skin fibroblasts. Under conditions that are routinely used for the preparation of cells for immunocytochemistry, this monoclonal antibody does not react with acid -glucosidase but in contrast with components of the cytoskeleton. Double-labelling experiments with the monoclonal antibody and rabbit anti-vimentin antiserum identified the cytoskeletal components as intermediate filaments. The implications of this observation for the use of monoclonal antibodies in immunocytochemistry in general are discussed.     

First visualization of dopaminergic neurons with a monoclonal antibody to dopamine: a light and electron microscopic study

A monoclonal antibody recently synthesized against dopamine (DA) was tested in rat and mouse brain sections after further treatment by PAP immunocytochemistry at the light and electron microscopic levels. Distribution of DA-immunoreactive cell bodies was examined in the substantia nigra (sn), the ventral tegmental area (vta), and the raphe nuclei. DA-immunoreactive fibers were investigated in two DA projection systems, the striatum and the septum. Many dopaminergic cell bodies were found in the sn and the vta. Some scattered DA neurons were encountered in the pars reticulata of the sn. The dorsal raphe and linearis raphe nuclei displayed sparse immunoreactive neurons and a dense plexus of DA fibers. Immunoreactive fibers were observed in the entire striatum, more dense in the ventral part. In the septum, immunonegative neurons were outlined by thin DA fibers in synaptic contact with their somata or dendrites. According to our observations, this DA monoclonal antibody seems to be a selective and sensitive tool for studying the dopaminergic neuronal circuitry at both histological and ultrastructural level.