Indirect Immunofluorescence Procedure Research Articles

Localization of an ecto-ATPase/cell-CAM 105 (C-CAM) in the rat parotid and submandibular glands.

We investigated an ecto-ATPase/cell-CAM 105 (C-CAM), previously shown to be distinct from the Ca2+ pump, in rat parotid and submandibular glands. Polyclonal antibodies raised against the enzyme were employed using indirect immunofluorescence, peroxidase-anti-peroxidase (PAP), and electron microscopic immunogold labeling procedures to visualize the location of the enzyme. In the PAP-stained sections and with immunofluorescence, labeling was observed on the luminal and lateral surfaces and the intercellular canaliculi of the acinar cells of both glands. The luminal surface of the intercalated ducts was brightly stained, whereas those of the striated and excretory ducts were less prominently labeled. The basal surface of the acinar cells in the parotid gland and the lateral and basal surfaces of the duct cells were not labeled. Apparent labeling was observed on the basal surface of the submandibular acinar cells. Electron microscopy revealed that for both glands the enzyme was primarily localized along the luminal border of the acinar cells, mainly in association with microvilli, with slightly less reactivity along the intercellular canaliculi and lateral borders and relatively little along the basolateral membranes. Gold labeling was also observed on the luminal borders of the intercalated and striated ducts. Possible functions of the C-CAM include breakdown of ATP, stabilization of the microvillar membranes, cell adhesion, and involvement in secretory mechanisms.

Somatostatin immunoreactive cells in lesional psoriatic human skin during peptide T treatment.

Peptide T has been shown to be an effective treatment in psoriasis. The mechanism through which peptide T works in psoriasis is at present unknown. Furthermore, a clearance of psoriasis has also been registered using the inhibitory peptide somatostatin. These observations all focus on the fact that peptide T, somatostatin, and/or other peptides, might provide a clue to understanding the etiology and pathogenesis of psoriasis. Therefore, the effect of peptide T administration on somatostatin containing cutaneous cell populations was investigated. Ten psoriatic patients were treated with peptide T (D-Ala-peptide T amide; 2 mg/day i.v.) for 28 days. Serial biopsies were obtained from the psoriatic lesions before, once weekly during and 4 weeks after discontinuation of the peptide T treatment. An indirect immunofluorescence procedure was performed using a polyclonal antiserum against somatostatin. Clinically, most of the patients responded successfully to the treatment. Immunohistochemical investigations of the serial biopsies revealed the appearance of extensive changes in the number of dermal somatostatin immunoreactive dendritic cells. We believe that peptide T may stimulate the local synthesis and/or release of somatostatin, or proliferation and/or migration of certain dendritic cell populations in psoriatic lesions during healing. Since the benefits of peptide T treatment of psoriatic patients parallel earlier investigations using somatostatin infusions, it is likely that somatostatin given exogenously or synthesized/released endogenously plays a vital role in inducing the healing process.(ABSTRACT TRUNCATED AT 250 WORDS)

T-Cell Receptor Diversity in Dendritic Epidermal T Cells in the Rat

The rat epidermis contains a population of dendritic CD3+ cells. For a better characterization of these cells and to investigate their relationship to epidermal lymphocytes of other species, we stained rat epidermal sheets using a variety of monoclonal antibodies against rat leukocyte differentiation antigens in an indirect immunofluorescence procedure. Additionally, we attempted to define their T-cell receptor (TCR) isotype at both the nucleic acid and protein level. Results obtained showed that the majority of the CD3+ dendritic epidermal cells are CD45+, CD2+, TCR alpha beta-, major histocompatibility complex class II-, Thy-1-, asialo GM1-, CD4-, CD5-, and CD8- lymphocytes. We further observed that, in contrast to the mouse system, the rat epidermis additionally harbors a small but distinctive portion of dendritic CD3+ cells that exhibit reactivity with an anti-pan TCR alpha beta monoclonal antibody. Our further finding that rat epidermal cells enriched for CD3+ lymphocytes express full-length C delta mRNA suggests that the vast majority of rat epidermal T cells carry surface-bound TCR gamma delta moieties. On the basis of these findings, one may speculate that the indigenous T-cell population of the epidermis is not necessarily programmed to uniformly express monomorphic TCR gamma delta molecules but, to effectively fulfill its role in host defense, is capable of adaptation to the specific challenges encountered by a given species.

Immunocytochemical localization of endogenous codeine and morphine

Experiments carried out by indirect immunofluorescence and unlabelled antibody enzyme procedures revealed the presence of morphine-like immunoreactive material in the perikarya, fibers, and terminals of neurons in different, discrete areas of rat and human brain. The monoclonal and polyclonal anti-morphine antibodies used do not distinguish between morphine and codeine. Endogenous morphine seems to be stored in neurons as the 3-ethereal sulphate conjugate. This possibility is supported by the finding that, although active uptake of [3H]morphine has not been detected in brain synaptosomes, long-term i.c.v. injection of the tritiated opiate results in the accumulation of radioactivity inside the same neurons in which the endogenous alkaloids have been detected. Finally, striatal slices exposed to high K+ concentrations showed a rapid disappearance of the morphine-like immunoreactive material from neurons, indicating that endogenous alkaloids are released from neurons by depolarization.

Immunohistochemical Differentiation of Gastrin and Cholecystokinin in Gastrointestinal Tract of Chickens

The presence of cholecystokinin (CCK) in the gastrointestinal tract of chickens has not been well demonstrated, although immunological and chromatographic techniques have shown the presence of intestinal gastrin-CCK-like factors. Recently, a new peptide, structurally related to mammalian CCK, but with a gastrin-like activity, has been isolated from the digestive tract of chickens. The objective of this work has been: 1) to study the presence of gastrin-CCK-like immunoreactivity (IMR) in the digestive tract of chickens; 2) to distinguish chicken gastrin from CCK; and 3) to establish their distribution using specific antibodies. Tissue specimens from the proventriculus, gizzard, pylorus, duodenum, jejunum, ileum, ceca, and rectum were studied using indirect immunofluorescence procedures. The antibodies used were: 1) an antibody specific against the C-terminal pentapeptide common to gastrin and CCK; 2) one specific against CCK-33; and 3) one specific against chicken gastrin. Their use allowed the differentiation of two cellular populations which showed different affinities for the antibodies, indicating the presence of a gastrin-like peptide in the antrum and another CCK-like peptide in the small intestine, with the highest concentration in the proximal ileum. Immunoreactivity was not found in any other studied area. Two different peptides of the gastrin-CCK family are present in the chicken’s gastrointestinal tract. However their differentiation and identification are more difficult than in mammals due to the greater structural similarities of these peptides in birds.

CULTURED EPITHELIUM ALLOGRAFTS

The effects of in vitro dendritic cell (DC) depletion on the survival of epidermal sheet allografts were studied in a murine model. Newborn (1-3 days old) mouse skin was used. Langerhans cell (LC) and Thy-1+ dendritic epidermal cell (Thy-1+ DEC) depletion was achieved using: (1) a prolonged culture period (7 days), or (2) the anti-IA and anti-Thy-1.2 mAbs followed by complement treatment. DC (LC and Thy-1+ DEC) depletion was assessed on sheets and cultured cell suspensions by an indirect immunofluorescence procedure. They showed that, after 7 days of culture or after the antibody-complement treatment, epidermal cultures were depleted of LC and Thy-1+ DEC. Cultured sheets were grafted onto the muscle of H-2-incompatible recipients. The control experiments were: (1) full epidermis and DC undepleted sheet allografts, and (2) DC depleted and undepleted sheet isografts. The full epidermis was totally rejected after 9 days. However, no rejection sign was ever seen in any of the isografts. The in vitro produced and allografted epithelia did not show any necrotic sign until the 11th day postgrafting. However, on days 12-13, the DC depleted allograft's color changed from pink to brown. On days 14-16, degradation of the allografts resulted in a complete denudation of the underlying muscle. Immunohistological analysis of the allografts revealed the presence of a monocyte and lymphocyte infiltration starting from the 11th day postgrafting, with the presence of polymorphonuclear leukocytes on day 14. These results suggest that LC and Thy-1+ DEC depletions were not sufficient to prevent allograft rejection.

An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies.

Indirect immunofluorescence procedures reported thus far are not effective at localizing two antigens in the same preparation when both primary antibodies are raised in the same species. In this case, the secondary antibodies can crossreact with both primary antibodies. We report here a protocol in which mouse monoclonal antibodies (MAb) specific for actin and myosin were used sequentially to stain the same frozen section of guinea pig skeletal muscle. The myosin-specific mAb was applied first and was localized with a rabbit anti-mouse IgG-rhodamine secondary antibody. The sections were then "blocked" with a non-binding mouse MAb and unconjugated goat anti-mouse IgG F(ab) fragments. The actin-specific mAb was then applied and localized with a rabbit anti-mouse IgG-fluorescein secondary antibody. Laser scanning confocal microscopy and image analysis demonstrated that the I-bands, the A-bands, and the H-bands of each sarcomere were clearly identifiable by this approach. This protocol is not limited to use with mouse MAb but can be easily modified to permit indirect immunolocalization of two antigens in the same sample using any pair of same-species primary antibodies.

Cocaine-mediated disruption of microfilament integrity within neural-fold neuroepithelial cells of chick embryos cultured in vitro

The effects of cocaine on microfilaments within neuroepithelial cells of neurula-stage chick embryos cultured in vitro were investigated by an actin indirect immunofluorescence procedure. Results of this study suggest that the cocaine-induced inhibition of neural-fold closure in chick embryos cultured in vitro reported previously is mediated by a loss of microfilament integrity within the neuroepithelium.

Cryptosporidiosis: multiattribute evaluation of six diagnostic methods

Six diagnostic methods (Giemsa staining, Ziehl-Neelsen staining, auramine-rhodamine staining, Sheather's sugar flotation, an indirect immunofluorescence procedure, and a modified concentration-sugar flotation method) for the detection of Cryptosporidium spp. in stool specimens were compared on the following attributes: diagnostic yield, cost to perform each test, ease of handling, and ability to process large numbers of specimens for screening purposes by batching. A rank ordering from least desirable to most desirable was then established for each method by using the study attributes. The process of decision analysis with respect to the laboratory diagnosis of cryptosporidiosis is discussed through the application of multiattribute utility theory to the rank ordering of the study criteria. Within a specific health care setting, a diagnostic facility will be able to calculate its own utility scores for our study attributes. Multiattribute evaluation and analysis are potentially powerful tools in the allocation of resources in the laboratory.

Monoclonal antibody specific to alpha-2-->3-linked deaminated neuraminyl beta-galactosyl sequence.

Fusion of spleen cells from a BALB/c mouse immunized with KDN alpha 2-->3Gal beta 1-->4Glc beta 1-->1Cer ((KDN)GM3) with P3-X63 Ag8.U1 (P3U1) mouse myeloma cells yielded a hybrid cell line that produced monoclonal antibody that bound to (KDN)GM3, but not to Neu5Ac alpha 2-->3Gal beta 1-->4Glc-beta 1-->1Cer ((Neu5Ac)GM3). The specificity of the monoclonal antibody was determined chiefly by the enzyme-linked immunosorbent assay procedure. This antibody was found to react most strongly with (KDN)GM3 and less strongly with a glycoprotein containing a number of KDN alpha 2-->3Gal beta 1-->3-GalNAc alpha 1-->3[8KDN alpha 2-->)n-->6]GalNAc alpha 1-->chains (< n > av = approximately 3). The results indicated that the monoclonal antibody (designated mAb.kdn3G) specifically and effectively recognized a disaccharide structure, KDN alpha 2-->3Gal beta 1-->, and specifically discriminated (KDN)GM3 from (Neu5Ac)GM3. The mAb.kdn3G was used to localize (KDN)GM3 in rainbow trout sperm by the indirect immunofluorescence procedure and the antigen was shown to be mostly, if not completely, associated with the external surface of the entire plasma membrane of rainbow trout sperm. The potential utility of mAb.kdn3G is addressed in searching for KDN-glycoconjugates which contain glycan units having the KDN alpha 2-->3Gal beta 1-->epitope structure.

An immunocytochemical study of putative neurotransmitters in the metacercariae of two strigeoid trematodes from rainbow trout (Oncorhynchus mykiss)

Whole mounts of the metacercariae of Diplostomum sp. and Cotylurus erraticus from rainbow trout have been treated cytochemically for the demonstration of cholinergic, serotoninergic (5-hydroxytryptamine) and peptidergic elements in the nervous system. Antisera directed against four vertebrate (pancreatic polypeptide, peptide YY, substance P and peptide histidine isoleucine) and two invertebrate peptides (neuropeptide F and FMRFamide) were used in an indirect immunofluorescence procedure in conjunction with confocal scanning laser microscopy (CSLM). Of the seven antisera tested, all except peptide histidine isoleucine showed significant immunoreactivity. Cholinergic and serotoninergic staining was found primarily in the central nervous system (CNS) and in cell bodies associated with the ventral and dorsal nerve cords in both trematodes. Peptidergic immunoreactivity was localised in the CNS and PNS of both genera, revealing an extensive innervation within the holdfast organ and in and around the oral and ventral suckers.

Rabbit ductus arteriosus during development: anatomical structure and smooth muscle cell composition.

The anatomical structure as well as the smooth muscle cell (SMC1) composition of the ductus arteriosus (DA) were studied in rabbits ranging in age from 29 days of gestation to 20 days after birth. Computer-assisted, three-dimensional reconstructions of hematoxylin-eosin stained serial cryosections from ductus arteriosus-aorta (DA-AO) junctures revealed that DA in animals near term is separated from the aorta by a "septumlike" structure that is continuous with the aortic wall. Two days after birth, obliteration of DA is almost complete, and a small "pocketlike" cavity appears in the pre-existing site in which DA merged into the aorta. This small cavity in the aortic arch was still evident in the large majority of animals examined even 20 days after birth, as also demonstrated by scanning electron microscopy. At this time period DA consisted of a central, fibrotic region surrounded by several layers of SMC (the ligamentum arteriosum, LA) and ended within the aortic media just above the small cavity, forming a round "scar." Vascular SMC composition of DA during closure was examined by means of indirect and double immunofluorescence procedures, using a panel of monoclonal antibodies against some cytoskeletal and cytocontractile proteins (vimentin, desmin, smooth muscle (SM), and nonmuscle (NM) myosin isoforms). "Intimal cushions" were particularly evident from 5 hr after birth and were found to be desmin-negative, homogenously reactive for vimentin and NM myosin, and heterogeneously stained with anti-SM myosin antibody. In SMC subjacent to the "intimal cushions," distribution of vimentin and SM myosin was homogeneous, whereas the one of desmin and NM myosin content was heterogeneous. The cytoskeletal and cytocontractile protein content displayed by SMC during the closure of DA is similar to that of "intimal thickening" found in some pathological conditions of the arterial wall in adult rabbits. Completation of DA closure (day 2) was accompanied by the disappearance of cellular heterogeneity in myosin isoform distribution in both the "intimal cushions" and the underlying media. These results give new insights into: (1) the structure of DA-AO juncture, which can be relevant to the physiology of blood circulation in the fetus, and (2) the phenotypic similarity of vascular SMC populations involved in the formation of "intimal cushions" and "intimal thickening."

Analysis of islet cell antibodies reactivity to a human islet cell line.

A human pancreatic beta cell line (HP62) was tested for reactivity with islet cell antibodies (ICA) as compared with previously-established methods. Using indirect immunofluorescence test, we found that HP62 cell line failed to react in a specific way with ICA from type 1 (insulin-dependent) diabetic patients since sera from normal controls showed a reactivity similar to that found in the patients. So, the usefulness of this human beta cell line as a tool of immunological purpose is questioned when indirect immunofluorescence procedures are used.

Immunofluorescent labeling of centromeres for flow cytometric analysis.

A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody-labeled human and hamster chromosomes were 4-10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.

Insulin, insulin-like growth factors and glucose transporters: temporal patterns of gene expression in early murine and bovine embryos.

mRNA phenotyping by the reverse transcription-polymerase chain reaction (RT-PCR) method was used to compare the patterns of expression of insulin and insulin-like growth factor (IGF) ligand and receptor genes in preimplantation bovine embryos with those established previously for preimplantation murine embryos. In the early bovine embryo, transcripts for IGF-I, IGF-II and mRNAs encoding receptors for insulin, IGF-I and IGF-II were all detectable at all embryo stages from the 1-cell zygote to the blastocyst. In the mouse, IGF-II ligand and receptor mRNAs were not expressed until the 2-cell stage, and the insulin and IGF-I receptor mRNAs were not detectable until the 8-cell stage. Since transcriptional activation of the embryonic genome occurs at the 8- to 16-cell stage in the bovine embryo and at the 2-cell stage in the murine embryo, it is suggested that these transcripts are products of both the maternal and embryonic genomes in the bovine embryo whereas in the mouse they are present only after activation of the embryonic genome. Transcripts for insulin were not detected in preimplantation embryos of either species. Colloidal-gold immunocytochemistry with antibodies directed against the insulin receptor, IGF-I receptor and IGF-I ligand has confirmed the presence of these molecules in bovine blastocysts. RT-PCR and indirect immunofluorescence procedures demonstrated that the glucose transporter (GLUT) isoform 1 is present in murine embryos from the oocyte to blastocyst stage whereas GLUT 2 expression begins at the 8-cell stage.

Human acrosome biogenesis: immunodetection of proacrosin in primary spermatocytes and of its partitioning pattern during meiosis

Proacrosin biosynthesis timing during human spermatogenesis has been studied using the monoclonal antibody 4D4 (mAb 4D4). Frozen and paraffin-embedded sections of testicular biopsies were labelled by standard indirect immunofluorescence and avidin-biotin immunoperoxidase procedures. The labelling specificity was checked by immunochemistry assays on unrelated tissues and by western blotting of testis extracts showing that only the 50-55 x 10(3) Mr proacrosin was recognized by mAb 4D4. Proacrosin was first observed in the Golgi region of midpachytene primary spermatocytes. In late pachytene primary spermatocytes, proacrosin was observed in two regions located at opposite nuclear poles. During the subsequent steps of the first meiotic division, the two bodies containing proacrosin were located: (i) on opposite sides of the equatorial plate during metaphase; (ii) along the microtubular spindle during anaphase; and (iii) close to each chromosomal aggregate during telophase. Two bodies containing proacrosin were still observed in interphasic secondary spermatocytes. The single labelled area observed in early spermatids was found to increase considerably in size during spermiogenesis. Anomalies of proacrosin scattering were observed in patients with Golgi complex partitioning failure. These data reveal proacrosin biosynthesis during diploid and haploid phases of human spermatogenesis and the proacrosin partitioning pattern during meiosis.

Dual fluorescence combined with a two-color immunoperoxidase technique: A new way of visualizing diverse neuronal elements

A method is described that allows an estimation of the neurotransmitter-related immunoreactivity, morphology and relationship to other immunoreactive elements, of single functionally identified neurons in the central nervous system. First, neurons are identified electrophysiologically using intracellular recording and labelled by iontophoresis of lucifer yellow (LY). After fixation and sectioning of the brain tissue, the location of the labelled neuron is determined by fluorescence microscopy. Sections are then processed using an indirect immunofluorescence procedure in order to determine the antigen content of the labelled neurons. Antisera to LY and an avidin-biotin immunoperoxidase technique is then used to localize LY in a permanent form, while the other previously localized antigen is permanently visualised by using the fluorescent-labelled second antibody as a bridge antibody in a peroxidase anti-peroxidase technique. The method is illustrated by an examination of neurons in the medulla oblongata of the rat, that have been stained intracellularly with LY, their content of tyrosine hydroxylase assessed, and their relationship to other tyrosine hydroxylase immunoreactive neurons determined.

Acidic FGF and Other Growth Factors in Preretinal Membranes from Patients with Diabetic Retinopathy and Proliferative Vitreoretinopathy

The development and extension of fibrovascular or fibroglial membranes onto the retinal surface are a major cause of visual loss in diabetic patients with proliferative retinopathy and in patients suffering from retinal detachment with proliferative vitreoretinopathy. The pathogenesis of these proliferative diseases, however, remain poorly understood and the nature of growth-promoting mediators implicated in these phenomena has not been determined yet. Using indirect immunofluorescence procedures, three different growth factors known to be mitogenic for various cell components of preretinal membranes, acidic fibroblast growth factor, epidermal growth factor and insulin-like growth factor type I, were sought in 14 specimens of preretinal proliferative tissues. Similar results were obtained in diabetic preretinal membranes and tissues from patients with proliferative vitreoretinopathy. The three different growth factors were found diffusely in the connective stroma and around new blood vessels within the vascular walls. Some fibroblast-like and pigment epithelial-derived cells more markedly reacted with anti-growth factor antibodies. These results provide indications on the eventual involvement of three potent growth factors in intraocular proliferative diseases, but whether or not these mediators play an active role in the development of preretinal membranes remains to be determined.

Cell proliferation within the growth plate of long bones assessed by bromodeoxyuridine uptake and its relationship to glucose 6-phosphate dehydrogenase activity

Bromodeoxyuridine (BrdUrd) was incorporated into the DNA of proliferating chondrocytes of the tibiae and metatarsal growth plate of rats of differing ages. Immunodetection of the incorporated BrdUrd was achieved using a monoclonal antibody to BrdUrd and indirect immunofluorescence procedures. The labelling index within the growth plate was found to decrease with advancing age in the tibia and metatarsal. Glucose 6-phosphate dehydrogenase (G6PD) activity appeared to be related to chondrocyte proliferation as a significant correlation was observed between G6PD activity and the labelling index of the tibial chondrocytes. The results of this study indicate that G6PD activity is a possible marker for cartilage growth and also that the BrdUrd technique has several advantages over the conventional autoradiographic methods for the assessment of cell replication.

Regional differences in immunostainability of isolated metaphase chromosomes of Indian muntjac with anti-Z-DNA antibody

The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A3 R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.