Journal of Histochemistry & Cytochemistry

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.

High concentrations of glutaraldehyde (2-5%) were found optimal for fixation of glutamate. In the absence of glutaraldehyde, (para)formaldehyde does not permanently retain L-[3H]-glutamate or D-[3H]-aspartate previously taken up into brain slices. Rats were fixed by rapid transcardial perfusion with 2.5% glutaraldehyde/1% (para)formaldehyde, and brain samples osmicated, embedded in epoxy resin, sectioned, and exposed to specific antisera to glutamate (conjugated to carrier protein by glutaraldehyde), followed by colloidal gold-labeled second antibody. The gold particle density was higher over putative glutamatergic nerve terminals than over any other tissue elements (two to three times tissue average in cerebellum and hippocampus). Calibration by test conjugates containing known concentrations of fixed glutamate processed in the same fluid drops as the tissue sections indicated that the concentration of fixed glutamate in putative glutamatergic terminals in hippocampus CA1 was c. 20 mmol/liter. The grain density over the parent cell bodies was only slightly higher than the tissue average. (Grain densities over test conjugates of other amino acids, aldehyde-fixed to brain macromolecules, were similar to that over empty resin. Labeling was blocked by glutamate-glutaraldehyde but not by other glutaraldehyde-treated amino acids.) In other experiments, brain slices were incubated in oxygenated artificial cerebrospinal fluid (CSF) and then immersion-fixed and processed as above. Here, the ration of grain densities in putative glutamatergic terminals vs other tissue elements was greater than in perfusion-fixed material. Comparison of intra-terminal areas poor and rich in synaptic vesicles suggested that in this preparation vesicles contained at least three times the glutamate concentration of cytosol. In the glutamatergic synapses of the giant reticulospinal axons in lamprey the ratio was over 30. Prolonged K+ depolarization of hippocampal and cerebellar slices reduced the nerve terminal glutamate immunoreactivity in a Ca2(+)-dependent manner. The results suggest that glutamate is released by exocytosis at excitatory synapses and show that immunocytochemistry can be used to study the cellular processing of small molecules.

We studied the ultrastructure and the synaptic arrangement of glutamate-immunoreactive terminals in rats, in the superficial laminae of the spinal cord, the brainstem cuneate nucleus, and the thalamic ventroposterolateral nucleus, where a role for glutamate as neurotransmitter has been suggested by biochemical, physiological and pharmacological approaches. The antiserum employed was raised against glutaramate conjugated to keyhole limpet hemocyanin with glutaraldehyde, and was used for pre-embedding staining with an avidin-biotin-peroxidase method and for post-embedding staining with an immunogold procedure. Both methods yielded similar results, consisting of labeling of selected terminals in all the areas examined. Double immunogold labeling on the same thin section using antisera against gamma-amino-butyric acid (GABA) or substance P (SP), in combination with the anti-glutamate serum, showed that staining for glutamate and GABA was present in different terminals in all the regions examined; glutamate and SP were co-localized in a few terminals only in the superficial laminae of the spinal cord. By performing immunogold staining in combination with anterograde tracing, glutamate immunoreactivity could be localized in identified primary afferents to the dorsal spinal cord and cuneate nucleus, and in lemniscal afferents to the thalamus.

This study tested the hypothesis that the excitatory amino acid transmitters glutamate and/or aspartate are associated with the periaqueductal gray (PAG)-raphe magnus (NRM) projection. Retrograde neuroanatomical tracing procedures utilizing the tracers WGA-HRP or D-[3H]-aspartate were combined with immunocytochemical localization of glutamate or aspartate to determine if glutamate and/or aspartate immunostained neurons projected to the NRM. Both glutamate- and aspartate-immunoreactive cells in the PAG were found to project to the NRM. Double labeling immunocytochemichemical procedures indicated that glutamate and aspartate are co-localized in many PAG neurons, suggesting the following possibilities: (a) one of these two amino acids may serve as a precursor to the other; (b) both amino acids may be co-released from the same PAG neuron; or (c) both amino acids are present in high levels in the perikarya for metabolic purposes. At the EM level, both glutamate- and aspartate-immunoreactive terminals were identified in the NRM, strengthening the concept that both amino acids participate in synaptic transmission in this medullary nucleus. To determine if glutamate and aspartate are in fact released from PAG-NRM axons, the PAG was stimulated chemically with homocysteic acid (HCA) and amino acids were collected from the NRM using a microdialysis probe. Microinjection of HCA, but not vehicle, into the PAG resulted in the release of both glutamate and aspartate in the nucleus raphe magnus. These data suggest that both glutamate and aspartate are released from PAG fibers terminating in the NRM and provide strong support for the hypothesis that excitatory amino acids play a neurotransmitter role in the PAG-NRM pathway.

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.

Two derivatives of a tungstate cluster containing 11 tungsten atoms (W11PO39SiR4-) have been synthesized which enable them to be covalently attached to biomolecules at specific sites. The tungstate cluster is 1.0 nm in diameter, electron dense, and visible in the electron microscope. One derivative is a W11-sulfonyl chloride, reactive with amines and sulfhydryls. The second compound is a W11-thiosulfonate which can be used to label sulfhydryl groups. These new labels are beam resistant and provide significantly higher resolution then most other electron microscopy (EM) markers. Labeling of the protein albumin is described as an example.

A new class of membrane labels was synthesized which contain a tungstate cluster (having 11 tungsten atoms) and an aliphatic organo-tin moiety with various chain lengths (C4, C8, C12, C18, C22). These molecules were found to insert into synthetic phospholipid vesicles and biological membranes (human red blood cell membranes). The tungstate clusters can be individually visualized in the high resolution STEM or seen en mass in thin-sectioned labeled membranes in the CTEM. These new labels should provide a means for direct high-resolution imaging of lipid-phase systems.

An approach using autoradiography for the study of early changes in the distribution of water-soluble materials and the chemography involved was investigated. Radioactive calcium chloride (45Ca) was injected into the iliac vein of a rat. Ten seconds after the injection the rat was frozen in hexane (-90 degrees C). The frozen rat was embedded in 5% sodium carboxymethyl cellulose and blocked in the coolant. A sheet of plastic tape coated with a synthetic rubber glue was fastened to the trimmed block surface, and whole-body sections 2-10 microns thick were cut with a disposable microtome knife. Selected sections were freeze-dried and then covered with a dried autoradiographic emulsion film about 1 microns thick. The autoradiograph clearly showed the distribution of radioactive calcium in the calcification zone of long bones. The samples chosen to assess chemographic artifacts showed positive and negative chemographies on most of the tissues when these were kept at 23 degrees C, and although both chemographic effects were significantly reduced when the samples were kept at -20 degrees C, cells in several tissues still exhibited positive and negative chemographies. The technique can be used for the study of any animal whose size is suitable for whole-body freeze-sectioning.

We investigated the ultrastructure of blood group antigens A, B, and H in human eccrine glands by means of the immunogold labeling technique. Blood group antigens A, B, and H were found in the Golgi apparatus, secretory granules, and over the apical and basolateral cell membranes of dark cells of eccrine glands depending on the blood group phenotype of the donors. Both A and B antigens were found in the dark cells of AB donors. The labeling pattern of the Golgi stacks seemed to have a polarity whereby the anti-blood group A antibody labeled all the stacks, whereas anti-blood groups B and H bound to the trans side of the Golgi complex. These observations suggest that the blood group substances are secreted into the lumen after being processed through the Golgi apparatus and the immature and mature granules in the dark cells of human eccrine glands.

Evidence that the c-erbB-2 proto-oncogene is important in prognosis and oncogenesis in a number of human malignancies is increasing. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized to determine the amplification and protein expression of this proto-oncogene, respectively. These extraction techniques are often time consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Immunohistochemistry (IHC), on the other hand, is more often time and cost effective. In addition, IHC may offer enhanced sensitivity over extraction techniques because of the in situ nature of analysis. In data presented here, 71 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilution DNA hybridization and IHC, respectively. In 65 (92%) of 71 cases, high-level expression was associated with gene amplification, whereas moderate or low-level expression was associated with a normal diploid gene copy number. In five of the six discrepant cases, IHC predicted amplification which was not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. In 39 of the 71 total cases, Western immunoblotting was compared with IHC in the assessment of oncoprotein expression. Concordance was found in 33 (85%) of 39 cases. In four of the six discrepant cases, high levels of c-erbB-2 expression were demonstrated by IHC but not by immunoblotting. In these cases, intraductal disease and stroma-rich tumors again led to a relative paucity of neoplastic tissue within the specimens. We conclude that IHC offers a favorable alternative to either Southern analysis or Western immunoblotting in the assessment of c-erbB-2 gene copy number and expression levels of oncoprotein in human mammary carcinoma. Furthermore, IHC may prove advantageous to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

A polyclonal antibody (CL-B1/29) raised against a synthetic peptide with an amino acid sequence identical to the first 29 N-terminal residues of bovine bone-derived transforming growth factor-beta 2 (TGF-beta 2) was characterized and used for immunolocalization of TGF-beta 2 in adult mice. Reduced staining of immunoblots and tissue after absorption of the antiserum with the immunizing peptide or with TGF-beta 2 but not with purified TGF-beta 1 demonstrated that the reagent is specific for TGF-beta 2, with little or no crossreactivity with TGF-beta 1. The immunolocalization of TGF-beta 2 was investigated in formalin-fixed, paraffin-embedded cultured cells and murine tissue. Specimens pre-digested with testicular hyaluronidase demonstrated immunostaining predominantly of extracellular connective tissue matrix, whereas specimens pre-digested with pronase E demonstrated primarily cytoplasmic staining. Immunoreactivity was widely distributed in connective tissue, muscle, adsorptive and secretory epithelia, especially of endocrine tissue, and neural tissue of adult mice.

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

There is increasing evidence that autocrine- and paracrine-acting growth factors participate in cell and tissue development, maintenance, and renewal. Recent advances in histochemical techniques have facilitated the localization of growth factor messenger RNAs or polypeptides in tissue sections. However, the spatial relationships between the sites of growth factor transcription, translation, and post-translational processing to functional bioactive peptides have been difficult to correlate because each method of detection requires separate tissue sections. We undertook the simultaneous detection of epidermal growth factor (EGF) transcripts and EGF epitopes by combining immunohistochemistry methods with in situ hybridization. Adult mouse submandibular gland was chosen as a representative model because it contains sites of EGF biosynthesis which may participate in mediating the development, maintenance, and renewal of the organ through autocrine or paracrine mechanism(s). Granular duct (GD) cells demonstrated the presence of both EGF transcripts and EGF peptides. In contrast, the interstitial cells lying adjacent to glandular epithelium also contained relatively high levels of EGF transcripts, although no mature EGF peptides were detected. The experimental approach of co-localization and the resulting data indicate previously unreported sites of EGF transcription in glandular interstitial cells, which may provide molecular information required for the morphogenesis and differentiation of adjacent glandular epithelium.

Coexistence of neurotransmitters within single nerve fibers or terminals can be convincingly demonstrated by the use of multicolor immunofluorescence. The present study examined whether three-color immunocytochemical localization of coexisting neurotransmitters can be performed using the blue fluorophore AMCA. Spectrofluorometric examination of secondary antibodies conjugated with AMCA, fluorescein, and lissamine rhodamine showed that the peaks of excitation and emission were well separated and that dots of AMCA-conjugated IgG dried on slides were not visible when viewed using microscope filters for rhodamine and fluorescein. These findings suggest that AMCA might be suitable for three-color immunofluorescence. The usefulness of AMCA for triple labeling was tested directly by staining sections of rat brainstem and spinal cord for serotonin (5HT), substance P (SP), and either enkephalin (ENK) or prepro-thyrotropin-releasing hormone 160-169 (ppT), a marker peptide for thyrotropin-releasing hormone. Triple labeling for 5HT, SP, and ppT was observed in both brainstem and spinal cord but was only very rarely observed for 5HT,SP, and ENK. No evidence was found for artifactual triple labeling, although false negatives appeared to be possible in some circumstances. We conclude that AMCA can be combined with fluorescein and lissamine rhodamine for three-color immunofluorescent studies of coexisting neurotransmitters. In addition, the coexistence of 5HT with ENK appears to be much less common than the coexistence of 5HT with either SP or ppT.

In this work we used Allium cepa root meristem cells in actively growing conditions and under treatment with the protein synthesis inhibitors cycloheximide (CHM) and puromycin. Morphological and quantitative results indicate that these drugs induce dramatic alterations in nucleolar structure reflected by a decrease of nucleolar size, much more evident under treatment with CHM, and by segregation of its main components. Quantitative analysis shows a decrease in NOR-silver staining after treatment with CHM, whereas in cells treated with puromycin NOR-silver staining remains constant. Our results reveal a decrease in the Ag-NOR proteins under conditions of diminished cell activity, suggesting a direct relationship between the quantity of Ag-NOR proteins and transcriptional activity. Using two-dimensional gel electrophoresis and NOR-silver staining in gels, we have characterized some proteins corresponding to molecular weights of 28 and 31 KD and pI of approximately 5.2. After treatment with CHM, reactivity of these proteins against NOR-silver staining is diminished. By means of a morphological study, analysis of NOR-silver staining, and of anti-DNA and RNAse-gold labeling, we have tried an approach to the nucleolar organization in plant cells. Our results suggest that the fibrillar component shows a reticular distribution where fibrillar centers, as described in animal cells, are not distinguished.

By means of immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen, using specific antibodies raised against thymosin alpha-1 and against parathymosin. We observed prothymosin alpha immunoreactivity in lymphoid cells both in thymus and spleen. In the thymus, prothymosin alpha staining was more marked in cortex than in medulla. In the spleen, prothymosin alpha was found in lymphocytes of the periarteriolar lymphatic sheaths and was especially prominent in the germinal centers. Parathymosin immunoreactivity in the thymus was mainly localized in the medulla; positive cells were reticuloepithelial cells from the thymic reticulum and the blood barrier. Thymocytes were negative. In spleen, parathymosin was found in reticular cells arranged in a ring between the periarteriolar lymphatic sheath and the marginal zone. Our results do not support an exclusive role for these peptides as immune system hormones or cytokines.

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).

A combined alkaline phosphatase (AP) and dipeptidlypeptidase IV (DPP IV) staining reaction has demonstrated enzymatic heterogeneity of the arterial and venous segments of capillaries in rat skeletal muscle. This study compared the staining reactions of skeletal muscles in many commonly used laboratory animals, including the axolotl, chick, quail, Monodelphys, rat, mouse, hamster, guinea pig, rabbit, dog, monkey, and human. DPP IV activity was found in the venous ends of the capillaries and in the endothelium of some larger veins in many of the species but was never demonstrated in the arterial side of the circulation. AP was found in the arterial ends of capillaries in all species except the axolotl, and it was also found in the endothelium of larger arteries of most species. AP activity was absent in venous endothelium of all species except for birds and Monodelphys. DPP IV activity was found in the perineurium of intramuscular nerves of most species, and AP activity was commonly seen in tendons and intramuscular connective tissue. The interspecies variability found in this study shows that care must be taken in comparing experimental data involving this technique from one species to another, but within a species the technique allows a fine level of discrimination between functionally distinct compounds of skeletal muscle tissue.

The transport properties of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) were tested in the frog central nervous system. After delivery of the lectin to the lower brainstem by iontophoresis, stained axons and axon terminals, as well as neurons with richly arborizing dendrites, were observed indifferent regions of the brain and spinal cord even far away from the site of application. The large number and the Golgi-like appearance of labeled neurons situated rostral and caudal to the site of PHA-L deposit indicate that PHA-L is transported equally in both the anterograde and the retrograde direction in the central nervous system of the frog. This is in contrast with the mammalian nervous system, in which PHA-L is transported predominantly in the anterograde direction and the retrograde transport is poor.

We report the production of monoclonal antibodies (MAb) by an in vitro technique which react with principal cells of the renal collecting duct. Spleen cells were directly simulated in vitro with unsolubilized antigens, i.e., by direct contact with the apical site of cultivated principal cells or by contact with cell fragments. Out of several others two antibodies, IV1 and IV2, were selected, which specifically reacted with the principal cells of the collecting duct. MAbIV1 also reacted with Type A intercalated cells, indicating the existence of a common antigen in the apical membrane of both cell types. Type B intercalated cells were consistently unreactive. All other parts of the uriniferous tubule were also unreactive. In Western blot analysis MAb IV1 showed immunoreactivity with a 40 KD and a 43 KD antigen. Our experiments demonstrate the possibility of producing antibodies against unsolubilized antigens by a simple in vitro technique. The activity of particular lymphocyte in this in vitro system is shown by the specificity of the antibodies.

Immunocytochemical localization of DAMP, a reagent used to detect low pH intracellular compartments, was studied in cultured neurons from rat hippocampus and in frog retinas. We find that DAMP is more sharply localized and that the immunocytochemical reaction is stronger when horseradish peroxidase or other proteins are included in the medium used to administer DAMP to the cells. A likely explanation is that the proteins enter acidified endocytic compartments and there provide sites to which DAMP molecules can be attached during fixation.