Conventional Histochemical Stains Research Articles

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches.

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 µm from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

The immunity function of rodlet cells in the intestine of Binni fish (Mesopotamichthys sharpeyi).

Objective:Rodlet cells produce secretions of glycoproteins in nature. This study investigated the microscopic morphology, histochemical and immunohistochemical reactions, and distribution of the rodlet cells in the gut of Binni fish (Mesopotamichthys sharpeyi).Materials and Methods:Thirty samples were obtained from the cranial, middle, and caudal portions of Binni intestine immediately after being euthanized, fixed in Bouin’s solution for 18 h at 24°C, and had undergone routine histological processing, different conventional histochemical stains, and immunostaining with TNF-α and S100 protein antibody.Results:The intestine of Binni fish showed different stages of rodlet cells classified into three distinctive forms: vesicular, granular, and mature cells. Rodlet cells are poorly stained with hematoxylin and eosin. Their secretory granules have a weak positive reaction with periodic acid-Schiff (PAS) and Alcian blue (AB), and react positively to combined AB and PAS. Rodlet cells were stained lightly with Safranin O, observed pink in color by Giemsa stain, and showed reactivity to Masson’s and Mallory trichrome stains. Rodlet cells were immunostained positively against TNF-α and S100 antibodies, indicating that they have an immune function.Conclusions:Rodlet cells, with their neutral glycoprotein secretions, play a crucial role in the immunity of Binni fish intestine.

Prediction of mismatch repair deficient biliary tract cancer: Role of morphological features and host immune response detected by routine hematoxylin-eosin staining.

The objective of this study was to determine the frequency and predictors of biliary tract cancer (BTC) with deficient DNA mismatch repair (dMMR) in Japan. Immunostaining and microsatellite instability analysis were performed for mismatch repair-related proteins in tissue specimens from 662 patients who underwent surgery for BTC between 2001 and 2017 to identify dMMR-BTC. We compared dMMR-BTC and proficient MMR (pMMR)-BTC based on patient demographics, pathological features, and host immune responses characterized by the percentage of stromal tumor infiltrating lymphocytes (sTIL percentage) and tertiary lymphoid structures (TLS). The incidence of dMMR-BTC was 2.3%. Significant predictors of dMMR-BTC were its primary lesion being intrahepatic cholangiocarcinoma (odds ratio [OR] 6.34, P=.004), presence of signet ring cell component (OR 35.62, P<.001), sTIL percentage ≥40% (OR 3.43, P=.038), and presence of TLS (OR 22.22, P<.001). The sensitivity, specificity, and negative likelihood ratio for any one or more of these four variables to be positive were 93.3%, 57.8%, and 0.12, respectively. Evaluation of histopathological findings and host immune response based on conventional histochemical staining is useful for efficient and inexpensive diagnostic screening of dMMR-BTC patients.

Glycopattern analysis of acidic secretion in the intestine of the red-eared slender turtle; Trachemys scripta elegans (Testudines: Emydidae).

The secretion of the goblet cells in the intestine of Trachemys scripta elegans was studied in situ by histochemical methods to analyze the diversity of sugar chains, with particular regard to the acidic glycans. Conventional histochemical stains (Periodic acid-Schiff, Alcian Blue pH 2.5, High Iron Diamine) and binding with ten FITC-labelled lectins combined with chemical and enzymatic pre-treatments were used to characterize the oligosaccharidic chains. The intestine can be divided into three regions, i.e. a duodenum, a small intestine and a large intestine. Goblet cells were observed in all the three tracts and presented an acidic secretion. WGA, LFA, PNA and SBA binding was observed only after desulfation. Glycans secreted by the three tracts consist mainly of sulfosialomucins with 1,2-linked fucose, mannosylated, glucosaminylated and subterminal galactosyl/galactosaminylated residuals. Differences among tracts are quantitative rather than qualitative, with sulfated, galactosaminylated and glycosaminylated residuals increasing from duodenum to large intestine, and galactosylated and fucosylated residuals showing an opposite trend. Variation is observed also between apices and bases of villi in both duodenum and small intestine, where sulphation decreases from the base to the apex and glycosylation shows an opposite trend. Functional implication of these findings is discussed in a comparative context.

Chemiluminescence-based quantification of the colonization rates of Lotus japonicus roots by arbuscular mycorrhizal fungi

ABSTRACTSimple evaluation of the levels of root colonization by arbuscular mycorrhizal (AM) fungi is challenging for conventional histochemical staining because most roots show an endogenous background signal. Herein, we report a chemiluminescence-based novel method for the quantification of the relative colonization rates of AM fungi. Lotus japonicus seedlings were grown in two types of soils with different inoculum potential [i.e., the former vegetation was maize (host) or buckwheat (non-host)]. Indigenous AM fungi colonizing roots were labelled with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), which specifically targets the N-acetylglucosamine polymers of fungal cell walls. After the roots were removed from the soil and rinsed with water, all procedures were conducted in 24-well plates. AM fungi were detected by chemiluminescence in the presence of enhanced chemiluminescence (ECL) reagents, and the signal strengths were measured by image analyses. Post-staining of AM fungal structures in the presence of 3,3ʹ-diaminobenzidine (DAB) and H2O2 (DAB staining) revealed that even small colonization events (e.g., hyphopodia) could be detected, whereas the background signals observed in DAB staining were eliminated in the chemiluminescence method. Chemiluminescence thus provides both high sensitivity and high specificity. The chemiluminescence-based colonization levels of roots grown in maize soil were significantly higher than those of roots grown in buckwheat soil, demonstrating the feasibility for high-throughput quantification of the relative colonization rates of L. japonicus seedlings by AM fungi.

The predictive value of Gen-Probe's amplified Mycobacterium tuberculosis direct test compared with culturing in paraffin-embedded lymph node tissue exhibiting granulomatous inflammation and negative acid fast stain

The diagnosis of granulomatous inflammation with possible tuberculosis (TB) infection in histopathology is often difficult. There is a need for a rapid and reliable diagnostic test. Thus, we evaluated the performance of the Mycobacterium tuberculosis direct (MTD) test in specimens with granulomatous lymphadenitis and negative acid fast stains. The M. tuberculosis direct (MTD) test by Gen-Probe was performed on 45 formalin-fixed paraffin-embedded tissue samples including 34 lymph nodes. We measured the predictive values of the MTD test in specimens with granulomatous lymphadenitis and negative acid fast stains. The overall test sensitivity was 73.9%, and specificity was 95.4%. The MTD test sensitivity and specificity for lymph node tissue were 72.7% and 91.67%, respectively. In the presence of granulomatous inflammation, the MTD test sensitivity and specificity were higher than those for all tissue samples, at 75% and 100%, respectively. Based on this study, the MTD test should be used as a supportive test in addition to conventional histochemical or immunological staining methods when evaluating lymph node tissue with a granulomatous inflammation to deliver stronger evidence to support clinical decisions at a much earlier time than a culture would allow.

Abstract 4071: PAXgene Tissue: A new tissue fixation technology for simultaneous preservation of morphology and nucleic acids

Abstract Introduction: PAXgene Tissue (PAXgene) is a new, formalin-free fixation technology developed for simultaneous preservation of morphology and biomolecules in tissue samples. PAXgene fixed, paraffin embedded (PFPE) tissue is suitable for conventional histochemical and immunohistochemical staining as well as for extraction of high quality nucleic acids. Material and Methods: Cases of human lung, breast and colorectal cancer were divided and fixed in formalin, PAXgene, or snap frozen in liquid nitrogen (LN2). PAXgene fixed, stabilized specimens were stored for up to four days at room temperature prior to processing. Formalin fixed, paraffin embedded (FFPE) and PFPE tissue morphologies were compared using H&amp;E stain. RNA, miRNA and DNA were isolated from FFPE, PFPE and LN2 tissue. RNA expression was analyzed in 96-well RT-qPCR arrays with pre-designed assays, and miRNA expression profiling was performed with single Sybr-green or primer/probe based assays. DNA was analyzed by agarose gel electrophoresis, long-range, and multiplex PCR. Results: Morphology of PFPE samples was similar to or indistinguishable from FFPE tissue. A high correlation of both RNA and miRNA gene expression results was observed between PFPE and LN2 samples. For all PCR assays, Ct values were within +/− 2 Ct. In contrast, poor correlation was observed between RNA from FFPE compared to RNA from LN2 with differences of up to 10 Cts. Correlation of FFPE to LN2 was higher in the case of miRNA but was still not as high as that of miRNA from PFPE. High molecular weight DNA which performed well in PCR could be isolated from LN2 and PFPE tissue, but DNA from FFPE failed in long-range PCR and showed biased or no amplification in multiplexed PCR. Conclusion: The PAXgene Tissue fixation technology preserves morphology and nucleic acids in tissue samples. It enables morphological and multimodal research biomarker testing with one sample. PAXgene Tissue is for research use only. It is not intended for diagnostic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4071. doi:1538-7445.AM2012-4071

Honey as a substitute for formalin?

Formalin has long been the standard fixative for clinical routines worldwide. After the Formaldehyde Standard became law in the US in 1987, as a result of increasing concerns about the potential carcinogenicity of formaldehyde, attempts have been made to find safer alternatives. Alcoholic formalin is a useful fixative, because in addition to fixation, dehydration also is begun. For centuries, honey has been known to be an antibacterial agent with the potential to preserve compounds without harmful effects on its users. We compared the effects of honey fixation with other routine fixatives using conventional histochemical and immunohistochemical staining methods. Our results demonstrated that tissues fixed in either honey or alcoholic formalin and 10% neutral buffered formalin (NBF) have similar histomorphology. Honey fixation showed minor histomorphological differences among the various tissues; however, it did not influence affect correct diagnostic conclusions. Our results suggested that honey can be used as a safe alternative to formalin in histopathology.

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences.

Glycocomposition of the apocrine interdigital gland secretions in the fallow deer ( Dama dama)

The secretions of the tubular interdigital glands were investigated by conventional (Periodic-Acid Schiff, Alcian-Blue at different pH, Low Iron Diamine and High Iron Diamine) and lectin (Con-A, UEA-I, LTA, WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA) histochemical methods in adult males and females of different age of fallow deer during the breeding season. Sialidase digestion and deacetylation pre-treatment were also employed in conjunction with lectin histochemistry. The glandular epithelium consisted of a single layer of low columnar cells with typical apical protrusions. No substantial differences of the above histochemical staining in relation to sex and age were observed. Conventional histochemical staining revealed that the interdigital glands secreted neutral glycoproteins whereas acidic glycocomponents did not seem to be present. Lectin histochemical technique allowed us to disclose a great heterogeneity of glycoproteins with N- and O-linked oligosaccharides containing α- d-Man/α- d-Glc, GlcNAc, α-Fuc, terminal β- d-Gal-(1-3)- d-GalNAc, - d-Gal-(1-4)- d-GlcNAc, α-Gal and β-GalNAc residues. β-GalNAc and disaccharide β- d-Gal-(1-3)- d-GalNAc were also found as subterminal to sialyl moieties. The lack of sexual and age-related differences in the glucidic content of the glandular secretions seems to indicate that the glycoderivatives may play only an accessory role in the production of odoriferous signals in fallow deer.

Autofluorescence: A screening test for mycotic infection in tissues

Fungal infection is a major health concern as the clinical features are not very distinctive. Lack of rapid diagnostic techniques results in delay in diagnosis, which may even culminate in a fatal outcome. The fact that many pathogenic fungal organisms autofluoresce in hematoxylin and eosin (H and E)-stained sections under ultraviolet illumination led us to evaluate the role of autofluorescence as a rapid screening technique for fungal infections. The aim of the present study was to assess the value of autofluorescence as a screening method for detecting fungi on tissue sections and to compare the results of autofluorescence with conventional histochemical stains for fungi. Hematoxylin and eosin-stained slides of mycotic lesions were examined under fluorescent microscope and the findings were compared with results of Gomori's methenamine silver and periodic acid-Schiff stains. We found fungal autofluorescence in 63 out of 64 cases studied, with a sensitivity of 97.8% and specificity of 100% in comparison with fungal stains. This was statistically significant (P < 0.05). We conclude that autofluorescence can be used as a rapid screening method for identification of fungi in tissue sections as it does not require any other specialized staining procedure.

노래미, 쑤기미, 구실우럭 및 홍감펭 장관 점액질에 대한 조직화학적 연구

농어목에 속하는 경골어류 4종(노래미, 쑤기미, 구실우럭 및 홍감펭)의 장관 점액질의 조직화학적 성상을 밝히기 위해 peridic acid Schiff (PAS) 반응, alcialn blue (AB) pH 1.0 및 pH 2.5, AB pH 2.5-PAS, aldehyde fuchsin (AF) pH 1.7-AB pH 2.5 및 high iron diamine (HID)-AB pH 2.5 염색을 실시하였다. 장 점막에 있는 주름의 모양은 어종 및 부위에 따라 차이가 있었다. 장의 모든 부위에서 관찰되는 배상세포는 원주세포사이에 위치하고 있으며 그 모양은 구형이거나 타원형이었고 원위장에서 수가 가장 많았다. 구실우럭 중간장 및 원위장과 노래미, 쑤기미 및 홍감펭 장에는 산성점액질과 중성점액질이 나타났으며 산성점액질의 성상은 sialomucin이었다. 구실우럭의 근위장에는 중성점액질만 나타났다. 점액질 양과 성상은 장 부위에 따라 차이가 있었다. 쑤기미의 원위장은 산성점액질과 중성점액질 양이 비슷하였다. 노래미의 장관 모든 부위, 쑤기미의 근위장과 중간장, 구실우럭의 중간장과 원위장, 홍감펭의 원위장에는 산성점액질이 중성점액질보다 더 많았다. 홍감펭의 근위장과 중간장에는 중성점액질이 산성점액질보다 더 많았다. The conventional histochemical stains were used to study the properties of mucosubstances of the intestine in four teleostean species, i. e., Agramus agramus, Inimicus japonicus, Epinephelus chlorostigma, and Helicolenus dactylopterus, all belonging to the order Perciformes. The following methods were used; periodic acid Schiff (PAS), alcian blue (AB) pH at 2.5, AB pH at 1.0, AB pH at 2.5-PAS, and aldehyde fuchsin (AF) pH at 1.7-AB pH at 2.5 stain. The mucosal folds of intestines show differences in species and regions. Widely distributed in all portions studied, goblet cells situated between cylinderical epithelial cells are round or ovoid in shape. They were most densely distributed in distal intestine. In the middle and. distal intestine of Epinephelus chlorostigma and all regions of intestines of Agramus agramus, Inimicus japonicus, and Helicolenus dactylopterus, the presence of both acidic and neutral mucin was confirmed. The property of acidic mucosubstance was sialomucin. Neutral mucin was only encountered in the proximal intestine of Epinephelus chlorostigma. The amount and property of mucin showed differences in species and regions. In the distal intestine of Inimicus japonicus, the amount of acidic mucin is similar to that of neutral mucin. In all regions of intestine of Agramus agramus, proximal and middle intestine of lnimicus japonicus, middle and distal intestine of Epinephelus chlorostigma, and distal intestine of Helicolenus dactylopterus, acidic mucin occured more frequently than neutral mucin. The proximal and middle intestine of Helicolenus dactylopterus have more neutral mucin than acidic mucin.

Porcine Circovirus Type 2-Associated Cerebellar Vasculitis in Postweaning Multisystemic Wasting Syndrome (PMWS)-Affected Pigs

Porcine circovirus type 2 (PCV2) is associated with several syndromes in growing pigs, including postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome. In the present study, a previously undescribed neurovascular disorder associated with a PCV2 infection is described. Sixteen pigs showed clinical signs of wasting and neurologic deficits. Acute hemorrhages and edema of cerebellar meninges and parenchyma due to a necrotizing vasculitis resulted in degeneration and necrosis of the gray and white matter. Few to numerous PCV2 DNA and antigen-bearing endothelial cells were detected in affected areas of the brain using in situ hybridization and immunohistochemistry. Conventional histochemical stains, as well as the detection of caspase 3 activity and DNA strand breaks by the terminal transferase dUTP nick end labeling assay, showed numerous apoptotic endothelial cells in the vascular lesions observed. Sequencing of various brain-derived PCV2-specific amplicons revealed a strong identity between different isolates and an 89 to 100% identity to previous isolates. The phylogenetic tree showed that there was no clustering of isolates correlating to clinical signs or geographic distribution. This previously undescribed PCV2-associated neurologic disease has features of both postweaning multisystemic wasting syndrome and, to a lesser extent, porcine dermatitis and nephropathy syndrome. The available evidence suggests that direct virus-induced apoptosis of endothelial cells plays a role in the pathogenesis of this unusual PCV2-associated cerebellar vasculitis.

조피볼락, 용치놀래기, 송곳니베도라치 및 졸복 장관 점액질에 대한 조직화학적 연구

The conventional histochemical staining were used to study mucosubstances properties of intestinal striated border and goblet cells in four teleostean species, i. e., Sebastes schlegeli, Halichoeres poecilopterus, Bryzoichthys lysimus, and Takifugu pardalis. The following methods were used; PAS, AB pH 2.5, AB pH 1.0, AB pH 2.5-PAS, AF pH 1.7-AB pH 2.5 and HID-AB pH 2.5 stains. The mucosubstances of striated border in the proximal intestine and rectum of Sebastes schlegeli contained with neutral mucin, middle and distal intestine contained with neutral mucin and acid mucin. The striated border of all the intestines of Halichoeres poecilopterus contained with neutral mucin and acid mucin, and those of Bryzoichthys lysimus and Tnkifugu pardalis contained with neutral mucin only. The amounts of neutral mucin were moderate to considerable in Sebastes schlegeli and Halichoeres poecilopterus, minimal to small in Bryzoichthys lysimus and Tnkifugu pardalis. The amounts and properties in mucosubstances of intestinal goblet cells showed differences in species and regions. The intestinal goblet cells of Bryzoichthys lysimus, and Tnkifugu pardalis contained neutral mucin only while Sehastes schlegeli and Halichoeres poecilopterus contained mixture of neutral mucin, sulfomucin and sialomucin. The amounts of neutral mucin were considerable to large in distal intestine and rectum of Tnkifugu pardalis, while moderate to considerable in all intestines of Sehastes schlegeli, all the intestines except for middle intestine of Bryzoichthys lysimus, and proximal and middle intestine of Tnkifugu pardalis. Also it was minimal to small in middle intestine of Halichoeres poecilopterus. The intestinal goblet cells of Sehastes schlegeli contained mixture of minimal amounts of strong sulfmucin, weak sulfomucin and minimal to small amounts of sialomucin, and those of Halichoeres poecilopterus except for rectum contained mixture of minimal to small amounts of strong sulfomucin and sialomucin.

Complex carbohydrates occurring in the digestive apparatus of Umbrina cirrosa (L.) fry.

The glycosoaminoglycans in the digestive apparatus of immature fish have important biological functions and are involved in morphofunctional differentiation. The aim of this study was to investigate the glycoconjugate histochemistry in the different parts of the digestive apparatus (oesophagus, stomach, intestine) of Umbrina cirrosa (L.) fry using classical histochemical reactions (periodic acid-Schiff, Alcian blue pH 2.5, Alcian blue/periodic acid-Schiff, high iron diamine, low iron diamine) in conjunction with glycolytic digestions that degrade different classes of glycosoaminoglycans. No differences were observed in the reactivity to conventional histochemical staining of the oesophagus, stomach or intestine among 27-, 34- or 44-day-old fry. In the oesophagus, the mucopolysaccharides contained chondroitin sulphates B and A and/or C, heparan sulphate and chondroitin. In the stomach, only neutral glycoconjugates were revealed, whereas in the intestine there were only chondroitin sulphates. Some differences in the type and content of glycoconjugates were found in Umbrina cirrosa (L.) fry compared to those of adult subjects, probably related to different dietary habits and to changes in the environmental conditions.

Mineral Oil: The Best Xylene Substitute for Tissue Processing Yet?

A simple and effective method for the substitution of mineral oil for xylene in tissue processing is described. After a short dehydration with ethanol, tissue fragments are immersed in a mixture of ethanol, isopropyl alcohol, and mineral oil at 45°C and 50°C, with vacuum / pressure /agitation in order to complete the dehydration and initiate the infiltration that is finalized with mineral oil and melted paraffin wax in separate and consecutive steps.Using random samples from 38 different types of tissue, 92 pairs of slides were prepared, half from specimens processed with xylene and the other half with the new procedure. Each of the 92 pairs of slides, that included 12 different conventional histochemical stains and 21 different immunohistocliemical stains, was evaluated 9 times in a blind test by as many reviewers. The evaluation of the results with the X2 test showed that processing tissue with mineral oil is equivalent to processing tissue with xylene. Furthermore, mineral oil poses no health or environmental risks and has microtomy advantages as well. Since June199 this new procedure Ilas been the standard method for tissue processing at Mount Sinai Meclical Center of Greater Miami. (The J Histotechnol 23:143, 2000)

Glycohistochemical investigation of canine and feline zonae pellucidae of preantral and antral oocytes

Glycoconjugate modifications were analysed in the zona pellucida during development of oocytes in dog and cat using conventional histochemical staining methods with or without previous carbohydrate digestion. A series of lectins combined with desulphation and sialic acid degradation were applied. No differences were observed between dog and cat follicles using conventional histochemical staining methods. In both species, the zona pellucida and follicular fluid/intercellular matrix strongly reacted with PAS and high iron diamine stain (HID) and reacted moderately with low iron diamine stain (LID). Treatment with testicular hyaluronidase, chondroitinase ABC, chondroitinase AC and chondroitinase B treatment diminished HID and LID positivity of follicular fluid and intercellular matrix. Lectins that gave the most intense staining of the zona pellucida of both species were SBA, PNA, RCA-I, GSA-IB4 and WGA, indicating the presence of beta-D-GalNAc, D-Gal and GlcNAc residues. Sulpho- and asulpho-carbohydrates were identified in terminal and/or subterminal positions linked to sialic acid residues. In conclusion, the results indicate that glycosaminoglycans are not present in the zona pellucida of both species. Differences were observed in carbohydrate residues and in their spatial distribution, depending on species and developmental stage of the follicles. The similarity in lectin affinity between ooplasm and zona pellucida of oocytes present in follicles at different stages of development confirm the involvement of oocytes in zona pellucida production.

A new application of BCG antibody for rapid screening of various tissue microorganisms

Background: In routine dermatopathology there is growing demand for a simple, fast, cost-effective, and highly sensitive screening tool for the detection of microorganisms. Objective: Our purpose was to determine whether immunostaining with polyclonal anti– Mycobacterium bovis (BCG), which is known for its interspecies cross-reactivity, is a suitable screening method for many common microorganisms in dermatopathologic specimens. Methods: A total of 254 formalin-fixed, paraffin-embedded skin specimens of viral, protozoal, fungal, and bacterial infections were stained with appropriate histochemical stains and with anti-BCG. Results: Anti-BCG labeled bacteria and fungi with high sensitivity and minimal background staining, but did not react with spirochetes, viruses, or protozoa (Leishmania). The quality and sensitivity of anti-BCG staining were superior to conventional histochemical stains. Conclusion: Because of its cross-reactivity with many bacteria and fungi as well as its high sensitivity and minimal background staining, the anti-BCG immunostain is a promising screening tool for the detection of the most common bacterial and fungal microorganisms in paraffin-embedded skin specimens. (J Am Acad Dermatol 1998;38:56-60.)

Cytodiagnosis of amelanotic metastatic malignant melanoma: an immunocytochemical study.

Malignant melanomas are known to present diverse patterns and this can result in considerable difficulties for an interpretation of malignancy type from cytohistologic material. Such difficulties are further compounded, if melanin pigment cannot be demonstrated by conventional histochemical stains; unfortunately many cases do exhibit this feature, especially in its metastases. This study was designed to review cases of amelanotic metastatic malignant melanomas in a variety of cytologic samples which were received from 40 patients with a known history of malignant melanoma. Cytomorphologically, the cases were classified as classical, carcinoma-like, spindle cell type, lymphoma-like, and undifferentiated type (Table I). While in 36 of the cases, the diagnosis of metastases from a melanoma was confirmed based on an immunopositivity to a variety of melanoma markers (Table III), in four of the cases, the immunostaining indicated metastases from another primary source, which was subsequently found. Based on our study, we are of the opinion that an immunologic characterization is useful to conclusively diagnose the majority of cases of metastatic amelanotic malignant melanomas. Furthermore, we feel that a reliance on a single melanoma marker is not justified, and a panel of antibodies should be used to distinguish a metastatic amelanotic malignant melanoma from other metastatic neoplasms.

Immunohistochemistry for the diagnosis of aspergillosis in Turkey poults

Summary From each of two flocks (A and B) of poults comprising 14,100 females and 11,300 males, respectively, 15 poults were examined pathologically. Poults of flock A had signs of neurological disturbances whereas birds from flock B showed respiratory symptoms. Gross lesions were observed only in two poults from flock A in which minute circular areas of cerebral malaria were seen. Histopathologically, the brain lesions contained fungal elements, and so did some of the pulmonary granulomas detected in three and six poults out of four and six birds examined from flock A and B, respectively. Mycological cultivation was attempted from the brains and lungs of five poults from flock A. However, only from the brain of a single bird a fungus, identified as Aspergillus fumigatus, was grown. Immunohistochemistry was applied because the histomorphology of fungal elements within some lesions did not offer any characteristics allowing an assessment of the identity of the infective fungi. Moreover, as fungi could not be detected within all lesions, immunohistochemistry accomplished the screening of tissues. For immunostaining of tissues a panel of monoclonal and polyclonal antibodies identifying agents of aspergillosis, candidosis, fusariosis, scedosporiosis, and zygomycosis, was used. Due to a strong and uniform reactivity of all fungal elements with immunoreagents to Aspergillus spp. an unequivocal diagnosis of aspergillosis was established in all mycotic lesions. Apart from the establishment of an aetiological diagnosis, the application of immunohistochemistry also disclosed fungal fragments in granulomas which could not be identified with conventional histochemical stains.