ABC Technique Research Articles

Comparison between histochemical and immunohistochemical methods for diagnosis of sporotrichosis.

To compare the efficacy of histochemical and immunohistochemical methods in detecting forms of Sporothrix schenckii in tissue. Thirty five cutaneous biopsy specimens from 27 patients with sporotrichosis were stained by histochemical haematoxylin and eosin, periodic acid Schiff, and Gomori's methenamine silver methods and an immunohistochemical (avidin-biotin complex immunoperoxidase) (ABC) technique associated with a newly produced rabbit polyclonal antibody anti-Sporothrix schenckii. A total of 29 (83%) cases were positive by the ABC method used in association with anti-Sporothrix schenckii rabbit polyclonal antibodies. Histochemical methods, using silver staining, periodic acid Schiff, and conventional haematoxylin and eosin detected 37%, 23%, and 23% of forms of S schenckii, respectively. The ABC technique was significantly more reliable than periodic acid Schiff and silver staining techniques. It is concluded that immunostaining is an easy and rapid method which can efficiently increase the accuracy of the diagnosis of sporotrichosis in human tissue.

Immunohistologic demonstration of platelet-derived growth factor(PDGF) in skin biopsies from acute stage of Kawasaki disease

The involvement of endothelial cells and vascular smooth muscle cells in the initiation, formation and development of aneurysmal dilatation of coronary arteries in Kswasaki disease(KD) have been emphasized. It has been thought that specific growth factors may have an effect on anintimal thickening and vascular dysfunction. Plateletderived growth factor(PDGF) is reported to be involved in various clinical situations such as wound healing,atherogenesis,embryogenesis, and infectious diseases. In this regard, we explored the localization of PDGF in KD. Sections of skin biopsies from 8 KD patients were investigated using the mouse anti-human PDGF monoclonalantibody. Samples obtained in the acute stage were stained with ABC technique. As a result, PDGF was detected in the dermis and epidermis. The prominent staining for PDGF was found in the endothelial lining of dilated qapillaries and in the cytoplasm of macrophages~ Positive staining for PDGF was particularly associated with macrophage infiltrates. In conclus{on, it is postulated that PDGF may play an importantrole in the initiation and/or healing process of vasculitis, and skin lesion in KD. { 28

Evaluation of three techniques for differential diagnosis of prostatic needle biopsy specimens.

To determine whether acidic mucin staining, lectin histochemistry using Wisteria floribunda agglutinin, and immunohistochemistry using the monoclonal antibody EAB 903 are of benefit in distinguishing between hyperplastic and carcinomatous prostatic glandular tissue in needle biopsy specimens. Formalin fixed, paraffin wax embedded prostatic needle biopsy specimens of benign and malignant tissue were examined. Alcian blue-periodic acid Schiff staining was performed on 33 benign and 34 malignant cases. Wisteria floribunda agglutinin (WFA) binding sites were demonstrated by the avidin-biotin peroxidase (ABC) technique with and without neuraminidase pretreatment on 34 benign cases and 32 malignant cases. EAB903 anticytokeratin antibody binding sites were demonstrated using both an indirect immunoperoxidase (IIP) technique and an avidin-biotin peroxidase complex method on seven benign and 31 malignant cases. Acidic mucin staining was found in 17 of 34 malignant glands and was weakly positive in five of 33 benign glands. WFA positivity before neuraminidase pretreatment was present in 29 of 32 malignant glands and in 19 of 34 benign glands. After neuraminidase all benign and malignant cases showed positivity. EAB 903 positivity was seen in 11 of 31 malignant glands using the IIP technique and in two of 31 malignant glands using the ABC technique. In seven benign cases there was positivity in all glands using the IIP method with predominant basal cell staining in three and superficial cell staining in four. In benign cases using the ABC method two cases were negative. None of the three methods studied showed sufficient sensitivity and specificity to allow their recommendation for routine diagnostic use.

Proliferative Vitreoretinopathy: Lymphocytes in Epiretinal Membranes

To investigate the potential contribution of inflammatory and immune-mediated processes contributing to the pathogenesis of proliferative vitreoretinopathy (PVR), an immunohistochemical study was undertaken to characterize the infiltrating inflammatory cells in epiretinal membranes surgically removed from the eyes of patients with PVR. Twenty-one epiretinal membranes obtained surgically from eyes with PVR complicating rhegmatogenous retinal detachment were studied immunohistochemically using the ABC technique and a panel of monoclonal and polyclonal antibodies. T lymphocytes were found in 18 of the 21 specimens and generally constituted a small percentage of the total cell number. CD4+ T cells were found in 14 of the 18 membranes containing T cells. Three of six frozen membranes contained T cells that were positive for the interleukin-2 receptor. In 5 of 16 membranes studied, cells positive for the macrophage/monocyte marker were found. No B lymphocytes or neutrophils were identified, and there were no deposits of complement or immunoglobulins. Positive staining for the class II MHC antigen HLA-DR was found in 7 of the 21 membranes, a result that was more consistent in frozen than in fixed tissues. The study suggests that T lymphocytes are present in PVR epiretinal membranes and may be activated. These cells have the potential to play a role in the pathobiology of PVR.

Detection of surface antigen expression of pre- and post-cultured normal adult's bone marrow cells by using ABC technique and its significance.

The expression of surface antigens on both normal adult's bone marrow and multipotential progenitor cells (CFU-Mix) was detected by use of ABC technique. The culture system of CFU-Mix was an ideal model for studying the differentiation direction of hematopoietic cells. The monoclonal antibodies (McAb) CD3, CD4, CD8 specific for T-lymphocytic lineage, CD22 for B-lymphocytic lineage and CD11, CD13, CD14 for granulocytomonocytic lineage were all demonstrated in certain proportion in cultured CFU-Mix, which might be associated with the control of hematopoiesis. A decrease in the positive rate of OKT9 and CD34 in cultured cells might be related to the differentiation of hematopoietic cells. SZ-2 and SZ-21 could contribute to the identification of the presence of megakaryocytes in CFU-Mix.

Expression of cytokeratin and erbB-2 oncoprotein in Paget's disease of the nipple. An immunohistochemical study.

The occurrence of epithelial membrane antigen (EMA), low molecular weight cytokeratin (CK) and erbB-2 oncoprotein was investigated in twenty-eight cases of Paget's disease of the nipple (PD) to determine their diagnostic usefulness. The ABC technique with monoclonal antibodies was used on formalin-fixed, paraffin-embedded tissue. The Paget's cells showed positive immunoreactivity for all three antigens investigated in a high percentage of PD. Immunoreactivity for CK and erbB-2 oncoprotein was restricted to the Paget's cells, whereas EMA in some cases also stained the adjacent keratinocytes. Since CK and/or erbB-2 oncoprotein occurred in 93% of the cases, we conclude that demonstration of both antigens is useful in the diagnosis of PD. ErbB-2 oncoprotein was also found to be expressed in a high percentage of the underlying intraductal and invasive carcinomas.

An immunohistochemical study of phospholipase A 2 in peripheral nerve during Wallerian degeneration

Antiserum raised against purified Crotalus adamanteus venom phospholipase A 2 (PLA 2) was used to localise the enzyme in normal and crushed sciatic nerve to determine the effect of nerve trauma on PLA 2 distribution in the PNS. The tissue was Bouin's fixed, and stained immunohistochemically using the Vectastain ABC technique. Anti-PLA 2 antiserum showed weak reactivity with normal rat myelin, while only ‘resident’ macrophages stained in normal mouse sciatic nerve. Post-crush, in the mouse, myelin staining was localised close to the crush site, with strong lysosomal staining within both ‘resident’ and invading haematogenously derived macrophages. In the rat, myelin sheats stained strongly throughout the nerve very soon after crush, but no macrophages were detected using these antisera. Paranodal myelin and some Schmidt-Lanterman incisures were the first regions of the sheath to show an increase in PLA 2 staining post-crush, areas where the first ultrastructural changes occur in myelin after trauma, with staining of compact myelin closely following. Our findings support the idea that PLA 2 may be involved in the early process of myelin breakdown seen in Wallerian degeneration.

Distribution of calcium-activated neutral protease inhibitor in the central nervous system of the rat.

The ubiquitous existence of calcium-activated neutral protease (CANP, calpain), an enzyme whose activity is regulated by calcium ions and a specific endogenous CANP inhibitor (calpastatin), is well known. Although there has been much investigation concerning the distribution and role of CANP, investigations of the distribution of the CANP inhibitor using immunohistochemical techniques are rare. We made antiserum against a 40K fragment of cDNA corresponding to two C-terminal repeats of rat liver CANP inhibitor expressed in Escherichia coli. Using this antiserum, we examined the distribution of CANP inhibitor in the rat central nervous system by the ABC technique and compared it with the distribution of CANP. Neurons and glias were stained, with the cytosol stained diffusely and the cell membranes stained clearly and strongly. Axons and myelin were stained faintly, but nuclei and vessels were not stained. The distribution of CANP inhibitor was thus found to be similar to that of CANP.

Immunohistochemical and histopathological study of experimental rabies infection in mice.

An immunohistochemical and histopathological study using the ABC technique was carried out to examine time-sequential virus spread in the central nervous system (CNS) of mice after inoculation with the CVS strain of fixed rabies virus by different routes; intracerebral (ic), intraocular (io), intranasal (in), intramuscular (im) and subcutaneous (sc). Only the ic and io inoculations caused fatal infections, so that detailed analysis was conducted on mice inoculated by these two routes. In ic-inoculated mice, viral antigens were detected mainly in neurons in the cerebral cortex and in the pyramidal cells and granular cells of the hippocampus. After io inoculation, viral antigen was first detected in the trigeminal nerve ganglia, following which it spreads to the cerebral cortex and cerebellum. In the hippocampus only a few cells were viral antigen-positive at the early stage after io inoculation. There were no inflammatory lesions or Negri bodies in the CNS of mice infected by either route. This suggests that clinical signs such as ataxia or depression leading to death may be due to the direct effect of the virus on the functions of neural cells, but not to inflammatory reactions. The ABC method will be useful for the early diagnosis of suspected patients or animals to have the disease when conventional histopathological and immunofluorescent antibody techniques can not detect lesions or viral antigens.

IMMUNOHISTOCHEMICAL STUDY ON THE PATTERN OF 50 KD CYTOKERATIN IN PSORIASIS

Three biopsies of normal skin and 15 biopsies collected from patients with psoriasis vulgaris were analyzed for the expression of the 50 kd cytokeratin using direct immunofluorescence and ABC technique before and after local treatment with anthralin 0.1% in a petrolatum base, with 0.05% betamethasone dipropionate cream, and finally, after PUVA treatment. Antiserum against the 50 kd anti-cytokeratin reacted with tissue sections of normal skin, staining cells in the basal layer, while the psoriatic skin sections before the various treatments showed a staining concerning the whole thickness of the epidermis. After the various therapies, the 50 kd cytokeratin immunoreactivity was observed only in the basal layer of those psoriatic skin sections that showed complete clinical clearing, while it was observed in the whole thickness of psoriatic patches that did not clear. These data suggest that the normalization of the 50 kd cytokeratin expression pattern can be considered as a marker of clinical remission of psoriasis.

Immunohistochemical localization of protein kinase C isozymes.

We report the immunohistochemical localization of protein kinase C isozymes (types I, II, III and ζ) in various organs using the monospecific monoclonal antibodies: MC-la, MC-2a and MC-3a, and a polyclonal antibody for a synthetic polypeptide of ζ, reviewing results reported previously (2-4, 20, 21, 23), and discussing the technical aspect of immunohistochemistry on protein kinase C. For light microscopy, tissues were fixed and cryo-sections or paraffin sections were made. They were stained by PAP, ABC and immunofluorescent technique. For immuno-electrom microscopy, pre-embedding immunoperoxidase and post-embedding immuno-gold technique were done. By light microscopic analysis, tissue specific differential localization of isozymes was observed. Three isozymes (types I, II and III) were consistently localized in the cytoplasm of various kinds of cell. On the contrary, ζ-related protein was located in the nucleus of Purkinje cells. The increased staining for protein kinase C isozymes was observed in human thyroid cancer tissues and cultured glioma cells. By immuno-electron microscopy, diffuse localization of protein kinase C (type III) in the cytoplasm of rod bipolar cells of the retina was observed. Type II isozyme was densely localized in the hyaluromere of blood platelets, and translocated to the cell membrane by TPA administration. The immunohistochemical techniques were very effective in elucidating the tissue specific localization of protein kinase C isozymes and their pathological changes.

Extranodal oral lymphomas: Histologic subtypes and immunophenotypes (in routinely processed tissue)

Twenty-seven extranodal oral lymphomas were subclassified according to the National Institutes of Health International Working Formulation. Immunophenotypes were then determined by means of an ABC technique with newly generated antibodies that identify fixation-resistant antigens on lymphoid cells. Diffuse small and large cell lymphomas were the most frequently identified subtypes. B-cell-associated antibody, L26, stained a majority of tumor cells in all lymphomas. Although 4KB5 was a less consistent B-cell marker, it stained most lymphomas. Reactive T-cell infiltrates, identified with antibodies MT1, UCHL-1, anti-CD3, and OPD4, varied from slight to intense. MT1 occasionally showed cross-reactivity with neoplastic B cells. No “histiocytic” lymphomas were found, but reactive macrophage infiltrates were identified in many lymphomas with monoclonal antibody KP1. In view of the immunohistochemical results, all lymphomas were believed to be of B-cell origin. Although antibody panels of the type used in this study can be effective in subtyping routinely processed oral lymphomas, careful interpretation is required because of reactive T-cell infiltrates.

Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.

Hyperplasia of Somatostatin and Pancreatic Polypeptide Immunoreactive Cells in Dogs with Idiopathic Atrophy of the Exocrine Pancreas

The pancreatic tissue from 25 dogs with idiopathic atrophy of the exocrine pancreas, and from 6 control dogs, was studied histologically and immunohistometrically. Cells producing insulin (B), glucagon (A), somatostatin (D), and pancreatic polypeptide (PP) were identified using specific antisera and the ABC technique. Histometrical quantitation revealed differences in the distribution of these cell types between the right and left pancreatic lobe. Initial stages of atrophy showed little changes concerning the relative proportions of the four cell types examined. In more advanced stages of atrophy, however, there was significant increase in the percentage of the D cells in the cell population of the left lobe. B and A cells showed no significant changes. In final stages, only tiny tissue spots were considered a secondary and regenerative phenomenon, but an endocrine dysregulation cannot be excluded. Atrophy accompanied by diabetes mellitus and a lack of B cells seem to be due to a deficiency of insulin.

下咽頭癌および前癌病変のサイトケラチン発現性に関する免疫組織化学的研究

In order to obtain a more objective method to evaluate epithelial disorders and carcinomas of the hypopharynx, the expression pattern of cytokeratins (CKs) was investigated by ABC technique using several kinds of monoclonal antibodies that react monospecifically with each subclass of CKs. In normal epithelia, CK-19 was strongly positive in the basal layer but apparently reduced in suprabasal layers and completely negative in superficial layers, while CK-13 showed a striking contrast to CK-19, being expressed within the whole thickness of epithelia except only in the basal layer. These 2 subclasses were also observed in "abnormal" epithelia, and characteristic changes of their combination were demonstrated in proportion to the histological gradings. In invasive carcinomas, CK-19 was strongly positive in all carcinoma cells of poorly differentiated carcinomas. It was sporadically positive in moderately differentiated carcinomas, the more differentiated the more sporadic. It was completely negative in well differentiated carcinomas. CK-13, on the other hand, was negative in poorly differentiated carcinomas but positive in keratinized cells of moderately or well differentiated carcinomas. Strong expression of CK-1 was observed only in well keratinized cells of hyperkeratotic epithelia and well differentiated carcinomas. These characteristic findings are consistently observed in all samples and, then, may be useful in evaluating epithelial disorders and carcinomas of the hypopharynx, when used in conjunction with standard histological techniques. It seems most likely that these results play a part in investigating normal and abnormal processes of cell differentiation.

The Use of Improved Silver Impregnating Staining Method in the Differential Diagnosis of Tuberculoid Leprosy

A comparative study on the usefulness of Kawatsu's silver impregnating staining method compared with S-100 protein ABC technique for the differential diagnosis of tuberculoid leprosy was carried out. The results of neurohistological examination obtained from both methods were almost the same. It can be said that Kawatsu's method is useful and cost-effective, and thus suitable for practical use in developing countries.

Estimation of properties from conjugate forms of molecular structures: the ABC approach

The ABC technique for estimating properties of organic compounds from their molecular structures is presented, based on the contributions of Atoms and Bonds to the properties of Conjugates. Conjugates are alternative formal arrangements of valence electrons: a real chemical compound is a hybrid of conjugates. In ABC, all conjugates of a molecule are generated, and thermodynamic and quantum-mechanical properties are assigned to each conjugate, by summing contributions from atoms and bonds of the conjugate ; the properties of the compound are then obtained as combinations of the properties of all the conjugates. In a simple application, ABC estimates the heat of formation of alkanes within 2.8 kJ/mol, compared to 4.7 kJ/mol for group contributions. The ultimate goal is the estimation of fractional charges on individual atoms of a compound and electron densities of bonds, because there are related to intermolecular interactions and chemical properties

Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis*

The possible in vivo role of TGF-beta 1 in regulating various proteins of the extracellular matrix, including fibronectin, collagen I and III, and glycosaminoglycans, was examined by immunohistochemical methods during critical stages of lung morphogenesis in the 11- to 18-day-old mouse embryo. Sections of Bouin-fixed, paraffin-embedded whole embryos were exposed to polyclonal antibodies specific to synthetic peptides present in the precursor part of TGF-beta 1 (pro-TGF-beta 1), in the processed TGF-beta 1 (antibody CC), collagen I and III, fibronectin, followed by the PAP or ABC technique to visualize the location of the antibody. GAG were stained with Alcian Blue 8GX. Our results indicate colocalization of TGF-beta 1 expression and that of matrix proteins in the developing lung when branching morphogenesis (cleft formation) and tissue stabilization occur. The presence of TGF-beta 1 at the epithelial-mesenchymal interfaces of stalks and clefts at a time when matrix proteins can first be visualized in these areas, suggests a direct participation of the growth factor in the development of the basic architecture of the lung.

Lectin Histochemistry on Glycoconjugates of the Epidermis and Dermal Glands of Xenopus laevis (Daudin, 1802)

AbstractWe performed an investigation at the light microscopical level of the differential distribution of lectin‐binding sites among cells of the epidermis and glandular domains of the African clawed frog Xenopus laevis. Using a panel of biotinylated lectins (Con‐A. PSA, LCA, UEA‐I, DBA, SBA, SJA, RCA‐I, BSL‐I, WGA, s‐WGA, PHA‐E and PHA‐L) and an avidin–biotin–peroxidase complex (ABC), we have identified specific binding patterns.The results show that expression of saccharide moieties in Xenopus epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different sugar residues. Moreover, oliogosaccharides with “identical” biochemically defined sugar compositions can be distinguished. The method allowed further characterization of complex glycoconjugates of dermal glands. In view of these results, the ABC technique and the biotinylated lectins employed in the present study are believed to be a reliable method for the precise localization of saccharide residues of glycoconjugates present in ectothermic vertebrates.

Localization of two structural wall proteins in an insect spermatophore by immunoelectron microscopy

Spermatophores are acellular protein sacs which package sperm for transport. They are highly ordered structures derived from secretions that self-assemble in extra- cellular spaces. The mealworm spermatophore emerges from the male in an unexpanded state and consists of a multilayered fibrous outer wall, an organized inner core and a lumen containing semen. The spermatophore expands inside the female as its core is extruded and molded into new wall material. We are interested in the assembly and maturation of the spermatophore and have developed monoclonal antibodies which recognize epitopes of two different wall proteins, each secreted from a distinct cell type. We have employed immunoelectron microscopy (ABC Technique) to determine the location of these proteins in the wall and to examine their fate during the expansion process.Spermatophores were collected from copulating male mealworms (Tenebrio molitor). Some were allowed to fully expand. Samples were fixed in 1% formaldehyde and 0.5% glutaraldehyde in 0.1M phosphate buffer, pH = 7.2, for 2 h; dehydrated through alcohol and embedded in Epon.