Journal of Histochemistry & Cytochemistry

During embryonic development, and possibly also later in life, pancreatic islets of Langerhans originate from differentiating epithelial stem cells. These stem cells are situated in the pancreatic ducts but are otherwise poorly characterized. We found by immunohistochemical staining that protodifferentiated pancreatic epithelial cells from rat embryos of Day 13-Day 15 express the cytoskeletal protein keratin 20, similar to mature duct epithelium. During the period of islet morphogenesis, which occurs between Day 17 and birth, large aggregates of K20-positive duct cells were formed, which gradually differentiated into endocrine cells. This islet morphogenic mechanism has not been described thus far and we did not observe it in postnatal rats. During fetal islet formation, transient expression of vimentin was noted in the duct cells but not in endocrine cells. This intermediate filament protein is not observed in duct epithelial cells after birth. The proto-oncogene product bcl-2, a putative epithelial stem cell marker, was detected in duct cells from fetal and postnatal pancreas. We conclude that K20, vimentin, and bcl-2 are markets for the pancreatic (islet) stem cells.

Endothelin is a potent vasoconstrictive peptide first isolated from the supernatant of cultured porcine aortic endothelial cells. The purpose of this study was to determine the source of endothelin-1 (ET-1) production in rat uterus during pregnancy and to clarify its role in normal pregnancy. ET-1, as recognized by immunohistochemistry, was weakly expressed only in endometrial glandular cells in nonpregnant rats but was intensely expressed in both glandular and myometrial cells in the early postpartum period. In situ hybridization confirmed the localization of prepro-ET-1 mRNA in the cytoplasm of endometrial glandular and myometrial cells but not in stromal cells or in smooth muscle cells of blood vessels. Northern blot analysis detected prepro-ET-1 mRNA in myometrial tissue from pregnant but not nonpregnant rats. In particular, the expression of prepro-ET-1 mRNA was strongest in the early postpartum period compared with the various stages of pregnancy. These results indicate that, in addition to endothelial cells and endometrial glandular cells, myometrial cells also produce ET-1 and its production significantly increases in the early postpartum period. Therefore, ET-1 may play a pivotal role in controlling bleeding from the placental bed through myometrial contraction.

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

After partial hepatectomy, the liver is capable of complete restoration of normal hepatic size, architecture, and function (regeneration). To study roles of the extracellular matrix in regeneration, the temporal and spatial sequences of deposition of several components, including collagen I, III, and IV, fibronectin, laminin, heparan sulfate proteoglycan (perlecan), and chondroitin sulfate proteoglycans were characterized by light microscopic immunohistochemistry in rat liver after 70% partial hepatectomy. Consistent with previous reports, there was a brisk mitosis of hepatocytes after the partial hepatectomy. Of the extracellular matrix components studied, 1B5 epitope generated by chondroitinase ABC digestion on chondroitin sulfate proteoglycans exhibited the most dramatic changes; the epitope was detectable as early as 1.5 hr after partial hepatectomy and its immunoreactivity reached a maximum at 24 hr, then declined gradually. This transient expression of the 1B5 epitope was also detected in neonatal rat liver during development. By Western blotting, the 1B5 epitope was found on two forms of the core protein of chondroitin sulfate proteoglycans with apparent molecular masses of 163 KD and 152 KD, which were also regulated in the same temporal manner.

We used several approaches to assess the reliability and sensitivity of computer-assisted densitometry to detect regional changes in tissue antigen content as a function of immunohistochemical staining density. We designed a model system to mimic variations in antigen concentration in postfixed, slide-mounted rat brain sections by varying the ratios of conjugated (biotinylated) to unconjugated secondary antibody. Antigen concentration was also varied in tissue discs made from mixing rat brain homogenate with increasing amounts of tissue embedding compound. The monoclonal antibody bd-17 to the beta2/3 subunit of the GABAA receptor was used as the primary antibody. Immunostaining density was visualized with diaminobenzidine (DAB). There was a significant, positive linear relationship (r = 0.97-0.99) between immunostaining intensity and antigen concentration. With this approach, changes in antigen content of less than 10%, as reflected in immunostaining intensity, were detectable in brain sections. The low degree of variability in measures of regional variation in immunostaining in sections from naive rats (n = 7) suggested that the method was suitable for quantitative analysis and indicated the reliability of the method. This systematic study of the utility of computer-assisted image analysis for semiquantitative immunohistochemical analysis found the method to be both reliable and sensitive.

The four kinds of oligopeptides specific in amino acid sequence to a rat dopamine transporter (DAT), peptide-1-peptide-4, were chemically synthethized. An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method. Two monoclonal antibodies, MAbs H-1a and H-1b, were produced against one of the oligopeptides, peptide-1. Western blot analysis confirmed that the two antibodies recognized an approximately 85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also confirmed by an antibody absorption test using two synthetic oligopeptides, one of which is specific only to DAT. These results have confirmed the specificity of the present antibody to DAT. The expression and subcellular localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF). The antibody labeled the surface of PC12 cells. When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endoplasmic reticulum and then on the surface of growth cones from the beginning of neurite outgrowth. DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [3H]-dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT, blocked the [3H]-dopamine at a concentration of 10(-7) M. The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased.

We estimated the total number of calcitonin-immunoreactive C-cells in rat thyroid gland using the optical fractionator, the unbiased stereological method for estimation of numbers. It was necessary first to use a fixative composed of formalin, acetic acid, and ethanol to distinctly visualize the C-cells. The 40-microm-thick sections had to adhere to chromalum-gelatin-coated Superfrost Plus glass slides, and the immunostaining technique had to stain the C-cells evenly throughout the whole sections. Because the C-cells were irregularly distributed in the thyroid tissues, their counting required screening of about 500 fields per lobe, but the number of C-cells counted need not be high, about 90 per lobe. We estimated that rats have 185,000 +/- 42,000 C-cells (mean +/- SD; n - 7). The C-cell population did not differ significantly between the two lobes of a given rat, but it varied markedly among rats. The biological differences among the animals contributed 83% to the observed variability, whereas the methodological uncertainty contributed 17%. The serum levels of calcitonin and calcium were not closely correlated to the C-cell numbers. Our results indicate that variability in C-cell experiments can be reduced most effectively by increasing the number of animals used. However, the similar C-cell frequency found in the two thyroid lobes of each rat allows the use of one uniformly sampled lobe for quantification and the other lobe for further analysis.

A contemporary view of autoimmunity suggests that self-reactivity is a normal phenomenon, in contrast to the classical association between autoimmunity and immunopathology. We have previously demonstrated that monoclonal antibody SCH94.03, a natural autoantibody with polyreactivity towards several purified protein and hapten antigens, promotes central nervous system remyelination when passively transferred to SJL/J mice chronically infected with Theiler's murine encephalomyelitis virus, an established experimental model of multiple sclerosis. In this study we characterized the autoreactivity of SCH94.03 with endogenous mouse tissue using immunoperoxide and multiple-color immunofluorescence staining techniques on frozen tissue sections. Within the nervous system, SCH94.03 labeled fibrous astrocytes, ependymal cells, ganglion satellite cells, and a sub-population of microglia, oligodendrocytes, and peripheral nervous system neurons. Outside the nervous system, SCH94.03 labeled gastrointestinal tract smooth muscle and luminal epithelium, erythrocytes, and interdigitating dendritic cells in peripheral lymphoid organs. These data indicate that SCH94.03 is a multi-organ reactive autoantibody and support the hypothesis that autoantibodies can have a beneficial rather than a pathogenic function in central nervous system demyelinating diseases.

D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.

The most common route of transmission of HIV is via the mucosa. We compared human and macaque intestinal epithelia to determine whether the SIV macaque system can be used as a model to study HIV transmission by the rectal route. The overall morphology of the macaque gut mucosa is very similar to that of humans. Differentiation markers follow the same pattern as in the human system. The carcinoembryonic antigen (CEA) is apical in epithelial cells of the rectum and is absent from the small intestine. Blood group H antigen is expressed by enterocytes but not by colonocytes or rectocytes. Galactosylceramide, a potential alternative receptor for HIV in epithelial cells, is expressed in all intestinal segments as in humans. In absorptive cells it is apical and intracellular in the rectum, colon, and cecum, whereas it is only intracellular in small intestine. In goblet cells the galactosylceramide is present in intracellular vacuoles in all segments. It is also present on the membrane of mucous granules in colon and small intestine but not in rectum. We therefore believe that the macaque digestive tract may constitute a good model for the human digestive tract in the transmission of lentiviruses.

The tonsil crypt epithelium contains membranous (M)-cells that transport antigens from the lumen to underlying lymphoid cells, thereby initiating specific immune responses. Mechanisms mediating the adhesion of antigens to the M-cell surface are important for effective and selective uptake of potential pathogens but are still poorly understood. Therefore, the carbohydrates present on crypt epithelial cells of the rabbit palatine tonsil were studied by lectin histochemistry. Ultrathin LR White sections were incubated with a panel of eight lectins conjugated to colloidal gold or biotin. The glycocalyx of the apical membrane of M-cells was selectively labeled by UEA-I, LTA, HPA, and VVA, whereas that of the remaining squamous epithelial cells preferentially bound RCA-I and PNA. WGA and ConA showed only little binding, with no discernible preference for any of the cell types. Double labeling of UEA-1 together with anti-vimentin antibodies revealed that UEA-I-positive epithelial cells also contained the rabbit M-cell marker vimentin, and vice versa. The results show that a specific composition of glycoconjugates, which differs from that on squamous epithelial cells, is found on M-cells of the rabbit tonsil. The M-cell-specific glycoproteins and glycolipids could be selectively targeted by microorganisms that adhere to M-cells and enter the host along this pathway.

There is a growing trend towards the objective quantification of immunohistochemical staining. However, quantification has not been used previously to optimize the original published immunohistochemical methods. We present a quantitative method for analyzing immunofluorescence staining employing the Applied Imaging MAGISCAN image analysis system, which has then been used to optimize major aspects of the standard immunofluorescent staining protocols. The optimization process resulted in a method that increased specific staining up to fivefold over typical published protocols, with no increase in nonspecific staining. The method is extremely reproducible. For slides stained by a single experimenter in one batch on one day, the coefficient of variation between replicate means is 1.2%. The image analysis protocol gave a linear response with increasing antigen concentration, as determined by using purified antigen dried onto slides. The revisions to the standard protocol presented here can also be applied to nonquantitative staining. It will help users of immunofluorescence to maximize their staining and may enable the detection of previously undetected antigens.

Acetaldehyde, the first product of ethanol in hepatocytes, can react with protein to form acetaldehyde-protein adducts (APAs). Because it has been suggested that these adducts could be involved in the pathogenesis of ethanol-induced hepatic lesions and in fibrogenesis, we performed an ultrastructural immunohistochemical study to precisely define the cellular and subcellular localization of APAs. A preembedding technique of indirect immunoperoxidase was performed in liver biopsy specimens from eight patients with alcoholic liver disease, using a specific antiserum against APAs. In all specimens, APAs were detected in the rough endoplasmic reticulum, in some peroxisomes, and in the cytosol of hepatocytes. In four patients with steatofibrosis or cirrhosis, labeling of Ito cells was also observed. In these cases, the same staining pattern was observed in the cytoplasmic processes of myofibroblasts in areas of fibrogenesis. When isolated rat Ito cells were incubated in the presence of acetaldehyde, APAs were also detected in the cytoplasm. These results show that APA formation occurs in hepatocytes at the sites of acetaldehyde production. Detection of APAs in human and rat Ito cells strongly suggests that acetaldehyde can diffuse into Ito cells and bind to cytoplasmic proteins to form local APAs. Because Ito cells are the main effector cells of liver fibrosis, detection of APAs in these cells points to their possible involvement in liver fibrogenesis.

The N-acetyltransferase (NAT) phenotype is an important determinant of individual susceptibility to occupational bladder cancer. N-Acetyltransferases arc known to metabolize aromatic amine bladder carcinogens, but the functional significance of NAT expression in the target organ is unclear. To resolve this issue, polygonal antisera against purified recombinant enzymes and C-terminal peptides of human NAT Type 1 (NAT1) and Type 2 (NAT2) were generated. Western blot analysis of exfoliated cells from human urine, pig bladder homogenate, and human bladder tumor-derived cell lines showed that NAT1 was expressed in all three systems, whereas NAT2 did not appear to be expressed in the bladder. Immunohistochemical analysis of human bladder tumor sections indicated that well-differentiated tumor cells expressed NAT1, with the highest level of expression being found in the umbrella cells that line the bladder lumen. Poorly differentiated tumor regions appeared to express NAT1 at lower levels than did well-differentiated areas. These findings support the hypothesis that aromatic amines are metabolized in the bladder epithelium by NAT1.

Studies of transplantation biology rely on the detection of donor hemopoietic cells in transplant recipients. Traditionally this has been achieved through ex vivo techniques, including flow cytometric analysis of cell surface markers to detect cells expressing specific epitopes, histochemical detection of cytoplasmic proteins, and the detection of Y chromosome-specific sequences by DNA hybridization. Studies using congenic models, such as the Ly5.1/5.2 mouse, or the utilization of fluorescent dyes, such as PKH-26, have allowed more in-depth analysis of transplantation, beginning to address key issues such as cell homing through cell tracking and elucidation of the "stem cell niche." However, these methods are limited by labeling sensitivity, specificity, crossreactivity and, in the case of PKH-26 labeling, the number of cell divisions the transplanted cells can make before the signal disappears. We have developed a fluorescent in situ hybridization (FISH) technique that utilizes a murine Y chromosome-specific "painting" probe to identify in situ individual transplanted male cells in paraffin-embedded sections of female whole bone marrow while maintaining good morphological integrity. This method is highly sensitive and specific, labeling more than 99% of male cells and no female cells, allowing each transplant to be assessed at the individual cell level. The technique provides unique opportunities to follow the path taken by transplanted cells, both during homing into the marrow and through their maturation and differentiation into mature, functional hemopoietic cells.

The avian song control system is an excellent model in which to study the effects of gonadal steroid hormones on neural and behavioral plasticity. Several of the brain regions that control song behavior concentrate androgens and/or estrogens. Investigations of the distribution and regulation of androgen receptors have been limited by the lack of a reliable immunocytochemical method to detect androgen receptors in the songbird brain. We describe a protocol by which the PG-21 polygonal antibody to the rat androgen receptor can be used to label androgen receptor-containing cells in the songbird brain. By treating songbirds of several species with testosterone 90 min before sacrifice and by using relatively low concentrations (0.5 0.75 microg/ml) of PG-21 antibody to reduce nonspecific background staining, we were able to obtain strong specific labeling of cell nuclei in androgen-sensitive brain regions. This technique will facilitate the study of the role of androgens in mediating neural plasticity in the avian brain. Testosterone pretreatment may also facilitate the use of this antibody to label androgen receptors in tissues from a wide array of nonmammalian species.