Indirect Immunofluorescence Procedure Research Articles

IMMUNOCYTOCHEMICAL IDENTIFICATION OF ACTH CONTAINING CELLS IN THE HYPOPHYSIS OF THE CRESTED NEWT

SUMMARY Employing a specific antiserum, anti-ACTH, cells corresponding to Type III basophils and pars intermedia cells were stained (with both the indirect immunofluorescence and PAP procedures) in the hypophysis of the crested newt.

Evidence of interstitial Cajal-like cells in human gallbladder

The aim of this study was to assess the presence of interstitial Cajal-like cells (ICLCs) in the human gallbladder and to determine their distinctive characteristics on the basis of double immunohistochemical staining. Gallbladder specimens were obtained from 30 patients subjected to elective laparoscopic cholecystectomy for symptomatic gallstone disease. Tissue samples were fixed in 4% phosphate-buffered paraformaldehyde, processed, embedded in paraffin, and, after sectioning, routinely stained with HE. Tissue antigens were retrieved using the heat-induced epitope retrieval (HIER) method. For simultaneous visualisation of two antigens, an indirect double immunofluorescence procedure was applied. ICLCs were defined as CD117-immunopositive and tryptase-immunonegative objects. They were predominantly fusiform in shape with sparse branches that were visible in some sections. ICLCs were observed throughout the organ including the gallbladder's fundus, body (corpus) and neck, being most numerous in the corpus. The ICLCs were detected almost exclusively within the muscularis propria and they were arranged parallel to smooth muscle cells. The following subpopulations of ICLCs were observed: ICLC-IM (intramuscular ICLCs) localised between smooth muscle fibres forming one muscle bundle; and ICLC-IB (interbundle ICLCs) localised within the connective tissue separating smooth muscle bundles. Thus, the presence of ICLCs in the human gallbladder was clearly identified, demonstrated by double immunohistochemistry which was found to be a reliable method for differentiating ICLCs from mast cells.

Evidence of interstitial Cajal-like cells in human gallbladder

The aim of this study was to assess the presence of interstitial Cajal-like cells (ICLCs) in the human gallbladder and to determine their distinctive immunophenotypic characteristics on the basis of double immunohistochemical staining. Gallbladder specimens investigated in the study were derived from patients (8 males, mean age 51.9±10.7 y; 22 females, mean age 52.9±15.1 y) subjected to elective laparoscopic cholecystectomy for symptomatic gallstone disease. Tissue samples were fixed in 4% phosphate-buffered paraformaldehyde, processed and embedded in paraffin. Sections from gallbladders were routinely stained with hematoxylin and eosin. Tissue antigens were retrieved using heat-induced epitope retrieval (HIER) method. For simultaneous visualization of two antigens an indirect double immunofluorescence procedure was applied. Primary antibodies used in this study were rabbit anti-c-Kit (anti-CD117) and mouse anti-mast cell tryptase. ICLCs were defined as CD117-immunopositive and tryptase-immunonegative objects. They were predominantly fusiform in shape with sparse branches that were visible in some sections. ICLCs were observed throughout the gallbladder, including the fundus, body (corpus) and neck, although they were most numerous in the corpus. The ICLCs were detected almost exclusively within the muscularis propria and they were arranged parallel to smooth muscle cells. The following subpopulations of the ICLCs were observed: ICLC-IM (intramuscular ICLCs) localized between smooth muscle fibers forming one muscle bundle and ICLC-IB (interbundle ICLCs) localized within the connective tissue separating smooth muscle bundles. In the current study we confirmed the presence of ICLCs in the human gallbladder. Applied double immunohistochemistry was found to be a reliable method for identifying ICLCs and for differentiating them from mast cells.

Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum

Detection of antinuclear (ANA) and antineutrophil cytoplasmic (ANCA) antibodies is extensively used for establishing a diagnosis in patients with clinical features suggestive of autoimmune disorders. The most common methods for the identification of positive patients' sera for ANA or ANCA are indirect immunofluorescence (IIF) and ELISA-based procedures. Considerable effort has been made in developing simpler automated assays for routine laboratory use. Recently a commercially available microsphere-based fluorescent assay has been introduced for the detection of ANA and ANCA. The aim of this study was to compare this technology with routinely used IIF and ELISA procedures, in patients with a suggested autoimmune disorder. A highly significant correlation between ELISA procedures for specific antibodies and the microsphere-based assays were obtained for both ANA and ANCA as well as for extractable nuclear antigens ELISA screening, indicating that multiplex technology could replace individual ELISA tests for the measurement of specific autoantibodies. However, a low sensitivity for identifying IIF-positive cases was obtained for both ANA (58.0%) and ANCA (59.1%), although there was a significant correlation between the assays. In conclusion, our data show that a microsphere-based fluorescent assay may be a valid platform for the simultaneous determination of circulating individual ANA and ANCA autoantibodies. Furthermore, multiplexing technology offers several advantages that will probably make it an attractive tool in the future. Nevertheless, until further studies are conducted that determine the clinical performance of the multiplex technology, the initial screening of patients for autoantibodies with IIF is still considered necessary.

Partial Characterization of an Adenovirus-Like Virus Isolated from Broiler Chickens with Transmissible Viral Proventriculitis

Transmissible viral proventriculitis (TVP) was experimentally reproduced in specific-pathogen-free chickens using a homogenate of proventricular tissue obtained from TVP-affected commercial broiler chickens. Thin-section electron microscopy revealed intranuclear, approximately 70-nanometer (nm), adenovirus-like viruses (AdLV) within proventricular lesions. The AdLV, designated AdLV (R11/3), could not be propagated using various avian and mammalian cell cultures or by inoculation of embryonated chicken eggs by yolk, allantoic, or chorioallantoic membrane routes. However, AdLV (R11/3) was successfully propagated by amniotic inoculation of embryonated chicken eggs, with detection of the virus in proventriculi and intestinal contents of hatched 2-day-old chicks (8 days postinoculation). Virus propagation was evident in in ovo-inoculated chicks by (1) gross and microscopic lesions in proventriculi consistent with TVP, (2) immunohistochemical localization of AdLV (R11/3) antigens in proventricular epithelium, (3) thin-section electron microscopic detection of intranuclear, approximately 70-nm AdLVs within proventricular epithelium, and (4) negative-stain electron microscopic detection of extracellular, approximately 70-nm AdLVs in intestinal contents. Indirect immunofluorescence and polymerase chain reaction procedures that specifically recognize groups I, II, and III avian adenoviruses failed to recognize AdLV (R11/3). The findings suggest an etiologic role for AdLV (R11/3) in TVP and indicate that this virus is distinct from known avian adenoviruses.

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.

Antibodies against cerebral M1 cholinergic muscarinic receptor from schizophrenic patients: molecular interaction.

We demonstrated the presence of circulating Abs from schizophrenic patients able to interact with cerebral frontal cortex-activating muscarinic acetylcholine receptors (mAChR). Sera and purified IgG from 21 paranoid schizophrenic and 25 age-matched normal subjects were studied by indirect immunofluorescence, flow cytometry, immunoblotting, dot blot, ELISA, and radioligand competition assays. Rat cerebral frontal cortex membranes and/or a synthetic peptide, with an amino acid sequence identical with that of human M(1) mAChR, were used as Ags. By indirect immunofluorescence and flow cytometry procedures, we proved that serum-purified IgG fraction from schizophrenic patients reacted to neural cell surfaces from rat cerebral frontal cortex. The same Abs were able to inhibit the binding of the specific M(1) mAChR radioligand [(3)H]pirenzepine. Immunoblotting experiments showed that IgG from schizophrenic patients revealed a band with a molecular mass coincident to that labeled by an anti-M(1) mAChR Ab. Using synthetic peptide for dot blot and ELISA, we demonstrated that these Abs reacted against the second extracellular loop of human cerebral M(1) mAChR. Also, the corresponding affinity-purified antipeptide Ab displayed an agonistic-like activity associated to specific receptor activation, increasing cyclic GMP production and inositol phosphate accumulation, and protein kinase C translocation. This paper gave support to the participation of an autoimmune process in schizophrenia.

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities.

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.

Histone H4 acetylation and DNA methylation dynamics during pollen development

The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.

Gamma-tubulin during the differentiation of spermatozoa in various mammals and man.

The distribution of gamma-tubulin as a marker of microtubule organizing centres (MTOC) was studied during spermiogenesis in rodents and in rabbit, monkey and man. A polyclonal antibody directed against human gamma-tubulin was used both for indirect immunofluorescence (IIF) and post-embedding immunogold procedures. In all species, gamma-tubulin was detected in the proximal and distal centrioles of round spermatids. In elongating spermatids, gamma-tubulin was predominantly found in the pericentriolar material (PCM) of both centrioles and particularly around the adjunct of the proximal centriole. At this level, some labelling was also associated with manchette microtubules, but other parts of the manchette and the nuclear ring were never labelled. We propose a role for distal centriole gamma-tubulin in axoneme nucleation and centriolar adjunct gamma-tubulin in manchette nucleation. The disappearance of gamma-tubulin in mature spermatozoa indicates that sperm aster nucleation should be dependent on oocyte gamma-tubulin. Remnants of gamma-tubulin in some human spermatozoa suggest that paternal gamma-tubulin also could contribute to sperm aster formation.

Eosinophil infiltration and degranulation in normal human tissue.

Localization of eosinophil granule major basic protein by immunofluorescence permits recognition of both eosinophil infiltration and degranulation. Over the past decade and a half, our laboratory has shown that eosinophil infiltration and degranulation occur in many diseased tissues in humans; among normal tissues studied as controls, only the gut showed striking eosinophil infiltration and degranulation. Using an indirect immunofluorescence procedure for the detection of major basic protein, we extended our analyses of normal human tissues to include tissues from essentially all body organs; a total of 117 biopsy/autopsy specimens were analyzed. To determine whether the method of tissue procurement affected the level of eosinophil degranulation in the normal gastrointestinal tract, normal proximal jejunum from six patients was biopsied using either an endoscopic forceps or a scalpel at the time of elective surgery and examined by immunofluorescence. Spleen, lymph node, and thymus tissues showed eosinophil infiltration with scant evidence of degranulation, but the only organ showing both eosinophil infiltration and remarkable degranulation was the gastrointestinal tract. Eosinophil degranulation was significantly increased in specimens obtained by endoscopic forceps compared to those obtained by scalpel (P = 0.021). These results indicate that tissue procurement methods affect the degree of eosinophil degranulation in the gut. Thus, among normal human body organs, both eosinophil infiltration and appreciable degranulation consistently occur only in the gut.

Immunocytochemical methods to study the distribution of Orientia tsutsugamushi in Leptotrombidium (Acari: Trombiculidae) chiggers.

Immunocytochemical methods were developed and tested for their ability to detect the distribution of Orientia tsutsugamushi in paraffin sections of adult chiggers (Leptotrombidium imphalum Vercammen-Grandjean & Langston). Rickettsial antigen was detected by application of a simple direct or amplified immunocytochemistry procedure and an indirect immunofluorescent procedure. In the direct procedure alkaline phosphatase conjugation to the mouse polyclonal antibody to the Karp strain was followed by the HistoMark Red test system to detect rickettsial antigen. The amplification procedure used a similar method but used an unlabeled primary antibody followed by secondary biotinylated antimouse IgG, streptavidin-alkaline phosphatase, and the HistoMark Red test system. The immunofluorescent procedure included a biotinylated secondary antibody followed by addition of a streptavidin-FITC conjugate. Specific tissue tropisms in infected chiggers were observed in the salivary glands, nervous tissue, and ovaries of adult female mites in all procedures; however, nonspecific fluorescence of the chigger limited definitive identification of tissue tropisms with the indirect immunofluorescent procedure.

Biofouling of Groundwater Systems by Thiothrix spp.

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.

Glycoproteins expression in apical pathologic tissues: Clinical incidences

Indirect immunofluorescence was used for the localization of the primary collagens, fibronectin and laminin. Specimens were extracted from untreated teeth with periapical lesions from patients 20 to 30 years of age. An histological examination enabled the differentiation of granulomas and cysts, and 5 microns sections were used for the indirect immunofluorescence procedure. Antibodies against Type I, Type III, and Type V collagen and for fibronectin and laminin were obtained from glycoproteins of human cells. The antibody against Type IV collagen was prepared from Type IV collagen of beef retina. All the glycoproteins investigated were expressed in apical lesions. The intensity of the immunostaining appeared more positive at the external area compared with the center of the lesion. The type IV collagen was specific for the basement membrane of cysts. The immunofluorescence reactions of fibronectin and of laminin were similar in intensity in both granulomas and cysts.

MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42.

MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli.

Improved microscopic identification of Clavibacter michiganensis subsp. sepedonicus cells by combining in situ hybridization with immunofluorescence.

An oligonucleotide probe was selected from the 16S rRNA gene of Clavibacter michiganensis subsp. sepedonicus for specific in situ hybridization. The rhodamine-labelled oligonucleotide probe was used in conjunction with an indirect immunofluorescence procedure based on a specific monoclonal antibody detected with a fluorescein-labelled conjugate. Simultaneous labelling of bacterial cells with the oligonucleotide and antibody probes allows accurate microscopic identification of single cells when isolation and other methods of confirming bacterial identity are not possible.

Role of cell surface glycosaminoglycans of human T cells in human immunodeficiency virus type-1 (HIV-1) infection.

To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV-1 infection in human T cells, HIV-1 binding and infection were determined after treatment of T-cell lines and CD4+ T cells from normal peripheral blood mononuclear cells (PBMC) with GAG-degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV-1/IIIB to MT-4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage-tropic strain HIV-1/SF162 than that of the T-cell line-tropic strain HIV-1/IIIB. The binding of HIV-1/SF162 was about 100-fold less dependent on cell surface HS than HIV-1/IIIB. Human HTLV-I positive T-cell lines expressed more HS than HTLV-I negative T-cell lines or normal CD4+ T cells when stained with anti-HS mAbs against either native or heparitinase-treated HS. With the exception of endo-beta-galactosidase (endo-beta-gal), GAG-degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV-1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV-1 infection by facilitating HIV-1/IIIB binding to MT-4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV-1 binding inhibition, other GAG-like molecules may also be involved.

Comparison of rapid immunofluorescence procedure with TestPack RSV and Directigen FLU-A for diagnosis of respiratory syncytial virus and influenza A virus

A rapid immunofluorescence format requiring 20 min for completion was as effective as conventional indirect and direct immunofluorescence procedures for detecting respiratory syncytial virus and influenza A virus antigens in clinical specimens. Rapid immunofluorescence was more sensitive than TestPack RSV and comparable to Directigen FLU-A immunosorbent assays, which require 20 min for completion.

Immunostaining and interphase arrangement of field bean kinetochores.

More than 100 sera from patients with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were tested in order to detect antigenic nuclear components of the field bean Vicia faba (2n = 12). Kinetochores of mitotic chromosomes and prekinetochores of interphase cells from root-tip meristems were specifically labelled via an indirect immunofluorescence procedure by antibodies of one of these sera. In 44% of interphase nuclei in which centromeres could be identified, only half (6) of the number of expected prekinetochores (12) was detected, circumstantially indicating at least transient association of homologous centromeres. Some nuclei showed clustering of centromeres at one pole (Rabl configuration). In metaphase chromosomes, each sister kinetochore contained a fluorescent spot. Western blotting of field bean nuclear proteins revealed four antigenic proteins of 28, 30, 64 and 68 kDa.

Recovery of Giardia lamblia Cysts From Chairs and Tables in Child Day-Care Centers

More than 11 million children attend commercial child day care in the US. This number is expected to increase as more mothers of preschool children enter the work force. Infectious diseases are readily transmitted in child day-care settings,1-4 where children are in close contact with one another for approximately 50 hours per week. Diarrheal diseases are common in these settings. Infectious diarrhea can be transmitted by person-to-person contact1,2,5 and, possibly, by contact with fomites.1,3 Many food service surfaces including tables, kitchen counters, ware-washing sinks, and dinner plates in the centers are contaminated with bacterial levels that exceed public health standards.6,7 Giardia lamblia is a pathogenic intestinal protozoan that may produce an acute infection characterized by diarrhea and other clinical symptoms.8 It infects children in day care at a higher rate than the general population.2,5 Family members are at risk of acquiring G. lamblia transmitted from children attending child-care centers3 where infectious cysts of G. lamblia may be shed by infected symptomatic or asymptomatic individuals.1,5 Intervention strategies that rely on exclusion of infected children from day-care settings are expensive and do not control Giardia infections in the child day-care environment.4 We compared the use of a commercially available, indirect immunofluorescent procedure with direct microscopic examination to detect G. lamblia cysts in stools of children attending day-care centers. Because the detection of G. lamblia on environmental surfaces would show their potential for transmitting giardiasis, we used the immunofluorescent procedure to look for G. lamblia cysts on various surfaces. Cysts were removed by swabbing surfaces, recovered on membrane filters, and detected using the indirect immunofluorescent procedure on the filters.