Indirect Immunoperoxidase Procedure Research Articles

Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive nerve terminals and on glucocorticoid receptor-immunoreactive nerve cell nuclei in the paraventricular hypothalamic nucleus in adult and old male rats

The phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive nerve terminal profiles and the glucocorticoid receptor-immunoreactive nuclear profiles have been characterized in the parvocellular part of the paraventricular hypothalamic nucleus of the adult (3 month) and the old (24 month) male rat. The phenylethanolamine-N-methyltransferase-, neuropeptide Y- and glucocorticoid receptor-immunoreactive structures have been demonstrated by means of the indirect immunoperoxidase procedure and analysed in a quantitative way by means of morphometrical and microdensitometrical approaches using both semiautomatic and automatic image analysis. During aging there is (a) a marked reduction in the number of neuropeptide Y-immunoreactive profiles, a moderate reduction of phenylethanolamine-N-methyltransferase-immunoreactive profiles and a small reduction in the number of glucocorticoid receptor-immunoreactive profiles without a significant change in the evenness of distribution of such profiles as evaluated by means of Gini's index; (b) a loss of the significant correlation in the distribution of the glucocorticoid receptor- and phenylethanolamine-N-methyltransferase-immunoreactive profiles at the two most caudal levels analysed (A5150 and A5270 μm) while a significant correlation developed between these two distributions at a more rostral level (A5400 μm); (c) a substantial decline in the overlap area of the glucocorticoid receptor- and phenylethanolamine-N-methyltransferase-immunoreactive profiles at four out of five rostrocaudal levels analysed; (d) a marked reduction in the density-intensity of the neuropeptide Y-immunoreactive profiles and a small significant reduction in the density-intensity of the phenylethanolamine-N-methyltransferase-immunoreactive profiles without any associated changes in the intensity of the glucocorticoid receptor-immunoreactive profiles. Furthermore, three-dimensional reconstructions of the overall distribution of the glucocorticoid receptor-, phenylethanolamine-N-methyltransferase and neuropeptide Y-immunoreactive structures have been made in the paraventricular hypothalamic nucleus of the adult male rat. The present results indicate a reduction of neuropeptide Y- and phenylethanolamine-N-methyltransferase-immunoreactive nerve terminal profiles in the parvocellular part of the paraventricular hypothalamic nucleus during aging. These results may in part reflect a loss of neuropeptide Y-like peptides in phenylethanolamine-N-methyltransferase-immunoreactive nerve terminals of the paraventricular hypothalamic nucleus, favouring our view that during aging the modulatory peptides may be lost, leading to a loss of plastic responses in the aging brain. Furthermore, the results open up the possibility that the glucocorticoid receptor immunoreactivity in the parvocellular part of the paraventricular hypothalamic nucleus is resistant to the aging process. Disturbances in the hypothalmic pituitary adrenal axis in the aged brain may therefore be related instead to reductions of glucocorticoid receptor turnover and not to changes in the steady-state levels of the glucocorticoid receptor.

Antigenic phenotype of radial growth phase melanomas with or without a vertical growth phase portion.

The expression of 4 melanoma-associated antigens and of class I and II HLA antigens was investigated in 12 superficial spreading melanomas (SSM) and in 8 SSM with a vertical growth pattern portion (SS + NM) by the use of monoclonal antibodies and an indirect immunoperoxidase procedure. Monoclonal antibodies 225.28, 763.74, CL.203, VF19-LL217, Q5-13, W6-32 and anti-HLA-DR, were used. Each antigen was more frequently expressed by SS + NM on the whole than by SSM and also by the radial growth pattern portions of SS + NM than by SSM. Vertical growth pattern portions of SS + NM were not antigenically similar to radial growth pattern portions in the same tumors. The high frequency of antigen expression in radial growth pattern melanomas seems to be associated with the appearance of a more invasive cell population.

Distribution and synthesis of type I and type III collagens in developing mouse molar tooth root

The distribution and synthesis of type I and type III collagens in the mouse molar tooth root have been investigated by correlating light and electron immunohistochemical data. Purified rabbit antibodies were raised against mouse type I and type III collagens and indirect immunoperoxidase procedures were used. In these conditions, predentin, pre-bone, and pre-acellular cementum were intensely immunostained for type I collagen. Both optic and ultrastructural data confirmed the presence of type I collagen at the epithelio-mesenchymal junction, but Hertwig's basement membranes remained unlabelled. The odontoblasts including the short polarized ones, osteoblasts, some cells of pulp mesenchyme and the perifollicular cells possessed type I collagen immunoreactivity in the rough endoplasmic reticulum (RER), Golgi complex and the secretory vesicles. Type III collagen immunoreactivity was strong in the perifollicular mesenchyme, light in the pulp mesenchyme and absent from the epithelio-mesenchymal junction, the predentin, pre-bone and pre-acellular cementum. Intracellular immunolabelling was detected at the ultrastructural level in the perifollicular cells by a faint homogeneous peroxidase deposit in the RER cisternae. Finally, these results, compared with previous biochemical and morphological data, represent the first dynamic aspect of collagens distribution and synthesis in the mouse molar root development. In terms of cell differentiation, our data also suggest that type III collagen synthesis does not occur during the odontoblast process of differentiation.

Detection of cytopathic and noncytopathic bovine viral diarrhea virus in cell culture with an immunoperoxidase test

Bovine viral diarrhea virus (BVDV) antigen was detected in cell culture with an indirect immunoperoxidase (IP) procedure using a specific monoclonal antibody, and an avidin-biotin-peroxidase complex. Cytopathic and noncytopathic strains of the virus showed similar patterns of IP staining until 3 days post-infection. At six days post-infection, intensity of staining decreased in cell cultures infected with noncytopathic virus, but not with cytopathic virus. The IP procedure detected BVDV antigen in cells used to isolate virus from tissues of aborted bovine fetuses and peripheral blood lymphpcytes of adult cattle. Immunoperoxidase detected BVDV isolates from 10 of 44 cases of abortion of which seven of these were noncytopathic. Noncytopathic BVDV isolates from the peripheral blood lymphocytes of 7 of 65 animals were identified.

Immunocytochemistry of ovine sporozoan encephalitis and encephalomyelitis

An indirect immunoperoxidase procedure was developed to detect Sarcocystis spp meronts in paraffin wax sections of infected ovine tissues. Ten rabbits seronegative to Sarcocystis spp and Toxoplasma gondii were injected intradermally or subcutaneously over 2 months with varying quantities (150 to 750 μg) of purified soluble cystozoite antigen derived from ovine heart microcysts. Serum from each rabbit was tested against paraffin wax preparations of S. ovicanis cysts, second generation S. ovicanis meronts and T. gondii cysts. Sera from three rabbits reacted with S. ovicanis meronts without any cross reaction to T. gondii antigens. Toxoplasma gondii and Sarcocystis spp-specific sera were used to identify the sporozoa in six cases of naturally-occurring ovine encephalitis or encephalomyelitis. Sarcocystosis was diagnosed in four lambs and toxoplasmosis in one lamb. The generic identity of the sporozoan in the sixth case was not established as meronts reacted with both Sarcocystis spp- and T. gondii-specific sera. The study demonstrates the value of the immunohistological technique of visualizing sparsely distributed meronts in naturally-infected tissues and shows that sporozoa of the genus Sarcocystis and T. gondii, and possibly also sporozoa the identity of which is as yet unclear, are associated with encephalitis of sheep in Britain.

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.

Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method.

We describe a simple and effective method for inhibition of endogenous peroxidase activity in the immunoperoxidase technique. Specimens are pre-treated with a mixture of azide and hydrogen peroxide, which is then followed by an indirect immunoperoxidase procedure. Comparison studies showed no significant loss of antigenicity or morphological details by this pre-treatment. The method is most useful for evaluating cell-specific antigens on specimens that have abundant endogenous peroxidase activity, such as blood and bone marrow.

Prelesional events in atherogenesis: Colocalization of apolipoprotein B, unesterified cholesterol and extracellular phospholipid liposomes in the aorta of hyperlipidemic rabbit

The appearance and accumulation of apolipoprotein B and unesterified cholesterol in the lesion-prone areas of the aorta in rabbits with diet-induced hyperlipidemia were investigated by histo-, and cytochemical techniques. Apolipoprotein B was detected by an indirect immunoperoxidase procedure both in the light and electron microscopy. Unesterified cholesterol was revealed using filipin and tomatine as specific probes. In the prelesional stages of atherogenesis, before the appearance of any structurally detectable lesions, as demonstrated by bright-field and fluorescence microscopy, apolipoprotein B and free cholesterol accumulated progressively in the extracellular matrix of the subendothelial space. At ultrastructural level, extracellular phospholipid liposomes, unesterified cholesterol and apolipoprotein B concomitantly appeared and accumulated focally in the same areas. Apolipoprotein B was preferentially located on the outer surface of the free cholesterol-containing phospholipid lamellae of the extracellular liposomes. In the lesional stages leading to fatty streak formation, the extracellular liposomes, apolipoprotein B and unesterified cholesterol had also topographically a superimposed localization pattern. Intracellular apolipoprotein B and unesterified cholesterol were also colocalized in some intimal lipid-laden cells. In the prelesional stages of hyperlipidemia the prevalent localization of apolipoprotein B around individual unesterified cholesterol-rich extracellular phospholipid liposomes, progressively accumulating in the subendothelial space, suggests their possible origin from serum-derived lipoproteins.

Evidence for discrete alterations in central cardiovascular catecholamine and neuropeptide Y immunoreactive neurons in aged male rats and in genetically hypertensive male rats of the Lyon strain.

A computer-assisted morphometrical and microdensitometrical analysis has been performed on cardiovascular noradrenaline (NA), adrenaline (A) and neuropeptide (Y (NPY) neurons in adult and 24-month-old male rats and on hypotensive (LL), normotensive (LN) and hypertensive (LH) male rats of the Lyon strain using the indirect immunoperoxidase procedures. It was found that in NPY/phenylethanolamine-N-methyltransferase (PNMT) costoring neurons of the CI area of the rostral medulla oblongata NPY-like immunoreactivity showed a more marked reduction than the PNMT immunoreactivity. Furthermore, within the parvocellular part of the paraventricular hypothalamic nucleus. NPY immunoreactive nerve terminal profiles were much more affected than the PNMT immunoreactive profiles during aging as revealed by a marked reduction in the number of profiles and by a marked reduction of absorbency values in the microdensitometrical analysis. Thus, in the NPY/PNMT costoring neurons of the A C1 group of the ventrolateral medulla projecting, for example, to the hypothalamus, the peptide transmission line may have a special vulnerability to the aging processes which may contribute to the development of hypertension in old people in view of a vasodepressor role of many central NPY/PNMT neurons. An extensive morphometrical and microdensitometrical analysis of the various catecholamine (CA) cell groups of the medulla oblongata of the LL, LN and LH rats of the Lyon strain was performed. In a comparison between LL and LH rats the A2 cell group of the LH strain showed a trend for an increase in the mean tyrosine hydroxylase (TH) immunoreactive cell body area and the C3 group showed a significant increase in the number of PNMT immunoreactive profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of human peripheral blood leukocytes for mast cell tryptase.

Murine monoclonal and goat polyclonal antibodies against tryptase, the dominant neutral protease and protein component in secretory granules of human mast cells, were used to assess the presence of tryptase in peripheral leukocytes. Carnoy's fluid-fixed cytocentrifuge preparations of enriched populations of lymphocytes, monocytes, eosinophils, and neutrophils showed no reactivity with anti-tryptase antibodies by a sensitive indirect immunoperoxidase procedure. Dispersed human lung mast cells showed strong granular cytoplasmic staining with both antibodies, whereas only approximately 50% of the peripheral blood basophils detectable with Wright's stain were detected with anti-tryptase antibodies, and these showed a staining pattern that was faint, granular, and cytoplasmic at high concentrations of antibody. At lower antibody concentrations mast cell staining was still intense, whereas basophils were not stained. Extracts of neutrophils and lymphocytes of up to 90% purity had undetectable amounts of tryptase by an ELISA sandwich immunoassay, as well as undetectable enzymatic activity with tosyl-L-gly-pro-lys-p-nitroanilide (a sensitive substrate for tryptase) in the presence of soybean trypsin inhibitor. Extracts of basophil-enriched (6 to 50% purity) preparations contained 0.046 +/- 0.013 pg of tryptase per basophil by the immunoassay along with 2 X 10(-9) +/- 0.8 X 10(-9) U of tryptase-like enzyme activity per basophil, compared with corresponding values of 12 pg, 480 X 10(-9) U of tryptase per human lung mast cell. Thus very small amounts of tryptase are present in human basophils (approximately 0.4% of that found in mast cells), but not in other peripheral leukocytes.

Macrophage-like cells of the pannus area in rheumatoid arthritic joints.

Frozen sections of pannus tissue taken from the joints of patients with rheumatoid arthritis have been investigated using immunohistological methods to determine the distribution of subsets of macrophage-like cells in this area. A panel of monoclonal antibodies including reagents specific in normal tissue for interdigitating cells (RFD1), macrophages (RFD7), epithelioid cells, (RFD9), monocytes (UCHMI), and osteoclasts (263C), were used. Indirect immunoperoxidase and combination indirect immunofluorescence procedures revealed the phenotypes of macrophage-like cells in four histologically distinct areas of the tissue: the synovial lining layers, the deeper stroma, areas of perivascular infiltration, and the articular cartilage junction where degeneration was occurring. It was discovered that 80% of the lining cells and a majority of macrophage-like cells of the stroma, express the phenotype RFD1+ RFD7+ UCHMI+. Cells with a typical 'dendritic cell' phenotype (RFD1+ RFD7-) were only present in the perivascular infiltrates, while 'classic macrophages' (RFD7+ RFD1-) were the cells accumulating at the cartilage junction. No significant numbers of RFD9+ epithelioid cells were seen. 263C+ osteoclasts were present in small numbers distributed throughout the stroma but did not appear to be involved in areas of cartilage degradation. This cellular distribution in the pannus is compared with previous studies on the rheumatoid synovium proper. It is concluded that a distinct inflammatory reaction occurs in the pannus and that classic activated macrophages are the cells involved in cartilage degradation.

Heterogeneity in distribution and content of p53 in SV40-transformed mouse fibroblasts

The high level and intranuclear location of the cellular phosphoprotein p53 are usually regarded as invariant features of SV40-transformed fibroblast lines. During the development of improved methods for immunocytochemical detection of p53 using SVA31 E7 mouse fibroblasts, we have observed unexpected and marked variations in its distribution. Cells grown on plastic coverslips were fixed in acetone and the content and distribution of p53 and SV40 large T-antigen analysed by an indirect immunoperoxidase procedure using monoclonal antibodies PAb122 and PAb 248 for p53, and PAb416 for large T. First, we observed in all cultures an apparent reversal of intracellular compartmentalization, with strong cytoplasmic and absent nuclear/chromatin positivity in mitotic cells and young daughter cells. More importantly, for a short period, between 18–24 h after trypsinization and cell passage, we observed a marked overall reduction in detectable nuclear p53 content in all cells with both PAb122 and PAb248 antibodies. The first observation also held for SV40 large T-antigen, the second only for p53. These variations have important practical implications for the immunocytochemical analysis of cellular content and intracellular compartmentalization of p53. The biological implications of our findings are also discussed.

The compartmentation of glycolytic and gluconeogenic enzymes in rat kidney and liver and its significance to renal and hepatic metabolism

An indirect immunoperoxidase procedure has been used to demonstrate sites of glycolysis and gluconeogenesis in normal rat kidney and liver. In kidney, the gluconeogenic enzyme fructose 1,6-biphosphatase was restricted to the proximal tubular epithelium, while the glycolytic enzyme hexokinase predominated in more distal segments. Intense staining for the biphosphatase in proximal convoluted tubular brush borders suggests that reabsorbed substrates may be used directly at this site in renal gluconeogenesis. In view of the high phosphofructokinase and pyruvate kinase activities present in collecting ducts, their relatively low hexokinase activities and their relatively pale immunostaining for hexokinase indicate that glycolytic substrates which feed into the pathway subsequent to the initial phosphorylation step, rather than glucose, may be the major energy source for the rat renal papilla. Immunostaining in the liver was consistent with the metabolic zonation of liver parenchyma, in that glucokinase occurred mainly in perivenous regions and fructose 1,6-bisphosphatase in periportal areas. The presence of such metabolic zonation is difficult to reconcile with the widely held view that the majority of hepatic glycogen is derived directly from glucose. A model for hepatic glycogen synthesis is proposed which links the concept of parenchymal zonal heterogeneity with recent biochemical evidence concerning the 'glucose paradox' and with microscopical studies on the dynamics of glycogen deposition after refeeding.

Rapid important paper on the cellular localization and distribution of estrogen receptors in the rat tel- and diencephalon using monoclonal antibodies to human estrogen receptor

By means of indirect immunoperoxidase procedures using the biotin- avidin method in combination with monoclonal antibodies to the human estrogen receptor it has been possible to map out distinct populations of nerve cells possessing nuclear estrogen immunoreactivity in rat brain. High densities of strongly estrogen immunoreactive nerve cells were especially observed in the medial preoptic area and the bed nucleus of the stria terminalis but also in the magnocellular part of the arcuate nucleus, the ventral premammillary nuclei and in the area between the medial and lateral hypothalamus including the lateral component of the ventromedial hypothalamic nucleus. Similar results were obtained in the male and female adult brain. Following castration of the male and female adult rat, the nuclear estrogen immunoreactivity did not change its location but the degree of immunoreactivity was increased. Administration of 50 ?g/kg of estrogen benzoate in the castrated animals induced a marked disappearence of the estrogen immunoreactivity in the nerve cells in all regions analyzed. The results give further evidence for the existence of a selective population of estrogen receptor containing neurons in the female and male brain of adult animals and that the estrogen free receptor is associated with the nucleus. Upon activation the nuclear estrogen receptors appear to loose this immunoreactivity probably due to a change in the conformation of the receptor protein.

Ultrastructural immunocytochemical demonstration of HLA class I antigens in human pathological liver tissue.

Major histocompatibility complex products Class I (HLA Class I) antigens are not expressed on the surface of normal human hepatocytes but become so in pathological conditions. The purpose of this study was to specify the ultrastructural topography of HLA Class I antigens expression. Nine human liver specimens, known from light microscopic investigation to display membranous positivity for HLA Class I antigens, were processed for immunoelectronmicroscopy using monoclonal anti-HLA Class I in an indirect immunoperoxidase procedure. HLA Class I antigens were detected on the basolateral membrane of hepatocytes and bile duct cells; some cisternae of the endoplasmic reticulum were also positive. The membranes of normal bile canaliculi of hepatocytes and the apical border of bile duct cells were negative. In one case of presumably drug-induced cholestasis, abnormal cholestatic canaliculi displayed HLA Class I antigens. These results indicate that HLA Class I antigens are synthesized by the hepatocytes and bile duct cells and incorporated into the plasma membrane; the basolateral expression follows the pattern as in other polarized cells. The expression in cholestatic canaliculi suggests a disturbed polarity of the hepatocyte.

Improved immunohistochemical visualization of helper/inducer T-cells by the simultaneous application of two noncrossblocking monoclonal antibodies.

We have studied the intensity of staining of helper/inducer T-cells in lymph node and tonsillar tissue using two commercially available monoclonal antibodies (OKT4 and Leu3a) with the indirect immunoperoxidase method. Paracortical and mantle zone helper/inducer T-cells were easily visualized by both monoclonal antibodies, but T-cells in the follicular center, though stained by Leu3a, were hardly demonstrable by OKT4. Excellent staining was obtained in the indirect immunoperoxidase procedure by incubating the sections with a 1:1 mixture of the two monoclonal antibodies which gave bright staining of individual cells throughout the lymphoid tissue. Dilution of the primary antibodies by 1:200 did not affect the results. It is concluded that the simultaneous application of OKT4 and Leu3a as primary antibodies in the indirect immunoperoxidase procedure is the method of choice for the in situ demonstration of helper/inducer T-cells.

Immunocytochemical localization of glucocorticoid receptor in target cells.

Antiserum prepared against highly purified glucocorticoid receptor was used for immunocytochemical studies. Rat liver and pituitary were chemically fixed, and histological sections were examined for immunoreactivity by an indirect immunoperoxidase procedure. In liver, immunoperoxidase staining was observed in the nuclei and cytoplasm of most hepatocytes. Kupffer, endothelial, and bile duct cells were not significantly stained. Control sections treated with preimmune serum remained unstained; preadsorption of antisera with purified liver glucocorticoid receptor resulted in a significant decrease in immunocytochemical staining. In adrenalectomized rats, nuclear staining was markedly reduced, whereas in adrenalectomized rats treated with cortisol acetate, the density of nuclear staining was comparable to and often slightly higher than that in intact animals. In the anterior pituitary, numerous cells were immunoreactive; their nuclei, cytoplasm, or both were stained. Alternate histological sections to those stained with antireceptor antibodies were processed for the localization of ACTH. It was found that the number of anterior pituitary cells that stained with the antireceptor antibodies exceeded the number of corticotrophs. Cells of the intermediate lobe pituitary were devoid of staining, whereas cells in the posterior lobe were stained. The presence of immunoreactive glucocorticoid receptors in pituitary cytosolic fractions was biochemically confirmed by immunoadsorption studies with [3H]triamcinolone acetonide-receptor complexes. This morphological localization of glucocorticoid receptor in liver and pituitary tissues demonstrated that immunocytochemistry can be successfully used to study localization of the glucocorticoid receptor. The known lack of response of intermediate pituitary cells to glucocorticoid may be secondary to a very small number or even an absence of glucocorticoid receptors.

Early appearance of desmin, the muscle-type intermediate filament protein, in the rat embryo.

Antisera raised to desmin, the protein subunit of muscle-type intermediate filaments (IFs), were used to study by indirect immunofluorescence and immunoperoxidase procedures the early development of skeletal muscle in the rat embryo. The specificity of the antisera (Dahl D, Bignami A: J Histochem Cytochem 30:207, 1982) was confirmed by immune blotting on chicken gizzard extracts and purified antigen. Desmin-positive cells were first observed on day 12 by immunofluorescence and on day 13 by the immunoperoxidase procedure. Desmin immunoreactivity was not found in caudal somites in which the dermatome was present, i.e., somites where the dorso-lateral part had maintained its definite boundaries and epithelioid characteristics. Desmin-positive cells were observed within the myotome of cranial somites where the dermatome had disappeared. Compared to day 13, desmin-positive cells had extended ventrally on day 14, while on day 15, they were found in the skeletal musculature of the trunk and the limbs.

The binding of human IgM paraprotein from cases of polyneuropathy associated with benign monoclonal gammopathy to specific neurons of the rodent brain

Human sera from patients with IgM paraproteinaemia were screened for IgM binding to frozen sections of the rat cerebellum by the indirect immunoperoxidase procedure. Out of 18 patients with a benign IgM gammopathy and associated demyelinating polyneuropathy (PPN), 17 showed binding to the surface of specific neurons, i.e. those of the deep cerebellar nuclei and the intermediate cell of Lugaro. Examination of other regions of the mouse brain suggests that the reactivity is restricted to cells of certain nuclei of the sensory-motor system. The use of F(ab) 2 and monoclonal anti-idiotype antibodies demonstrate that the binding involves the antigen binding sites of the paraprotein. This reactivity indicates that some neurons have on their surface an antigen having an hapten in common with human myelin-associated glycoprotein (MAG) and the pi granules of human Schwann cells and furthermore is an additional serological characteristic of this group of patients. In addition 3 sera from the PPN group stained a particulate component in the cytoplasm of all medium-sized and large neurons. A similar staining was found with 7 out of 52 other unselected IgM paraproteinaemias.

Laminin in rat sciatic nerve undergoing Wallerian degeneration. Immunofluorescence study with laminin and neurofilament antisera.

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.