Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections

Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.

Neuronal Ceroid-lipofuscinosis with prominent chorea and without visual manifestations: a case report

A case of neuronal ceroid-lipofuscinosis (NCL) is reported in a 11-year-old girl, whose main symptoms were progressive dementia since the age of 4 years and choreic movements since age 10. Seizures, myoclonus and visual deterioration were absent and optic fundi were normal. A cerebral biopsy disclosed two basic types of stored substance in the cytoplasm of neurons: a) severely balloned nerve cells in cortical layers HI and V contained a non-autofluorescent material, which stained with PAS and Sudan Black B in frozen, but not in paraffin sections; ultrastructurally, these neurons showed abundant corpuscles similar to the membranous cytoplasmic bodies of Tay-Sachs disease and, in smaller amounts, also zebra bodies; b) slightly distended or non-distended neurons in all layers contained lipopigment granules, which were autofluorescent, PAS-positive and sudanophil in both frozen and paraffin sections; their ultrastructure was closely comparable to that of lipofuscin. Similar bodies were found in the swollen segments of axons and in a few astrocytes and endothelial cells. The histochemical and ultrastructural demonstration of large amounts of lipopigments allows a presumptive classification of the case as NCL. However, the presence of involuntary movements, the absence of visual disturbances and the unusual ultrastructural features place the patient into a small heterogeneous group within the NCL. A better classification of such unique instances of the disease must await elucidation of the basic enzymatic defects.

Heterogeneity of Ia antigen expression on myeloblastic leukaemias: correlation with stage of maturation defined by cytochemical markers.

The expression of Ia-like antigen (Ia) has been studied in 55 cases of acute myeloid leukaemia (AML) in correlation with the expression of both Sudan Black (SB) and naphthol AS-D chloroacetate esterase (NCAE) stains. Operationally the AML cases were divided into three groups using only NCAE expression on the leukaemic cells: the first group with early maturation stage (MS1) consisted of 30 cases with less than 10% NCAE positive cells (SB: 15-100%): the MS2 group of 14 cases with 10-70% NCAE positive cells (SB: 65-100%) and the MS3 group of 11 cases with 70-100% NCAE positive cells (SB: 89-100%). Ia expression was determined by complement-dependent cytotoxicity, immunofluorescence and immunoperoxidase methods. A similar high percentage (80%) of patients from both group MS1 and MS2 expressed Ia on the surface of 32-100% of the cells. Furthermore, individual comparison of all cases from these two groups showed no correlation between Ia, NCAE and SB expression. Only in the 11 cases from the MS3 group, which included nine cases of promyelocytic leukaemias, was there a correlation between very low expression of Ia antigen with the high NCAE expression. Thus, for AML with a low degree of differentiation the expression of Ia seems to be independent of conventional cytochemical markers of cell maturation.

Anatomia e histoquímica dos órgãos vegetativos de Siegesbeckia orientalis (Asteraceae)

Descreveu-se a anatomia dos órgãos vegetativos de S. orientalis, em estrutura primária, enfatizando a caracterização e histoquímica de suas estruturas secretoras. Folhas, caules e raízes foram fixados em FAA50 e em sulfato ferroso e estocados em etanol 70%. Cortes transversais e longitudinais foram submetidos ao azul-de-toluidina pH 4,0, ao vermelho-de-rutênio, à reação de PAS, ao reativo Xylidine Ponceau, ao Sudan Black B e ao reativo Dragendorff. Anatomicamente, os órgãos vegetativos de S. orientalis são semelhantes aos caracteres descritos para Asteraceae. As raízes laterais são triarcas e o caule, um eustelo. Ductos estão ausentes na raiz e presentes no caule e na folha. Estas estruturas são de pequeno diâmetro, sendo delimitadas por quatro a cinco células epiteliais. No caule, os ductos estavam presentes no córtex, próximos à endoderme e na medula; nas folhas, associados aos feixes vasculares, tanto para o lado do xilema quanto para o do floema. As folhas são dorsiventrais e anfiestomáticas. Três tipos de estruturas secretoras foram observados: ductos, hidatódios e tricomas glandulares. Os testes histoquímicos aplicados demonstraram a presença de compostos fenólicos e alcalóides nos ductos e, nos tricomas, de compostos lipofílicos e fenólicos. Esses resultados indicam a complexidade da secreção produzida pelas estruturas secretoras na espécie.

Ultrastructure of the intercellular protuberances in leaves of Paepalanthus superbus Ruhl. (Eriocaulaceae)

Mature leaves of Paepalanthus superbus exhibit intercellular protuberances between the inner periclinal walls of the epidermal and the parenchyma cells surface, as well as on the surface of the parenchyma mesophyll cells. These structures are mostly prominent around the parenchyma cells, forming a gel capsule-like structure. Histochemical tests with ruthenium red indicate the pectic nature of the intercellular deposits, with scattered lipidic inclusions as revealed by sudan IV and sudan black B. Ultrastructural analyses show a fibrillar matrix with scattered fimbriate and tubular structures, and a distinct margin delimited by a dense membrane-like structure. Our results suggest that the protuberances are derived from secretory activity, and are formed after the development of the intercellular spaces. For P. superbus this structure may represent an important cell wall specialisation, related with the adhesion and transport mechanisms between cells.

Morfo-anatomia do sistema subterrâneo de Calea verticillata (Klatt) Pruski e Isostigma megapotamicum (Spreng.) Sherff - Asteraceae

Várias espécies herbáceas do cerrado apresentam sistema subterrâneo espessado, esses podem ser de natureza radicular, caulinar ou mista. Esses sistemas muitas vezes possuem potencial gemífero, promovendo o rebrotamento de ramos aéreos após um período desfavorável do ambiente, como uma seca prolongada ou uma queimada. A verificação da natureza dos sistemas subterrâneos é de extrema importância para utilização correta da terminologia dos mesmos. O objetivo desse trabalho foi fornecer informações sobre a morfo-anatomia dos sistemas subterrâneos de Calea verticillata e Isostigma megapotamicum, com ênfase na formação de gemas caulinares. Os sistemas subterrâneos foram coletados em áreas do cerrado do Estado de São Paulo. As espécies apresentam sistema subterrâneo bastante complexo, com estrutura anatômica mista, lignificada, auto-enxertias de ramos e raízes e elevada capacidade gemífera, ambas espécies são providas de xilopódios e as gemas têm origem cambial. Foi verificada a presença de canais secretores em C. verticillata e em I. megapotamicum, originados a partir de células derivadas do câmbio vascular. O teste microquímico realizado com Sudan black B confirmou a natureza lipídica da secreção. Em I. megapotamicum foi verificada a presença de cristais de inulina sob luz polarizada. Os sistemas subterrâneos aumentam as chances de sobrevivência das duas espécies estudadas às condições adversas do cerrado, pois apresentam alto potencial gemífero, promovendo o rebrotamento de ramos aéreos durante a estação favorável.

Spermathecae of the mangrove crab Ucides cordatus: a histological and histochemical view

Ucides cordatus is the most commercially, important mangrove crab in Brazil. In spite of its economic importance, there are few studies of its reproduction, in particular the female reproductive system. The present study describes the histology and histochemistry of the spermathecae of U. cordatus. Adult females were can lit monthly from July 2004 through June 2005, at Iguape, State of São Paulo. The crabs were anaesthetized, and their spermathecae removed and fixed in Davidson's fluid, following the histological routine for paraffin. The slides were stained with HE, xylidine Ponceau, PAS, alcian blue (pH 1.0 and 2.5), Sudan black B and picrosirius-haematoxylin. Histologically, the spermathecae possesses a capsule of conjunctive tissue, rich in collalgen fibres, which surrounds the secretory columnar epithelium. In the lumen, individual sperm packets are not observed; the spermatophores are intermixed with the seminal fluid and secretions of the spermathecae itself. A large proportion of the free spermatozoids and spermatophores are arranged in homogeneous masses in the proximal part of the spermathecae. The secretion produced by the columnar epithelium appears to promote the movement of the gametes to the fertilization chamber, in a ventral position, allowing fertilization of the oocytes. Histochemically, the secretion produced by the columnar epithelium was strongly positive for neutral polysaccharides, positive for acid polysaccharides, and weakly positive for proteins and lipids. Tills secretion forms a glycoprotein matrix which is associated with maintenance of the spermatophores, which can remain stored for long periods.

Ultramorphology and histochemistry of fat body cells from last Instar larval of the Pachycondyla (=Neoponera) villosa (Fabricius) (Formicidae: Ponerinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Staining methods applied to glycol methacrylate embedded tissue sections

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. on the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues.Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections.The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA. (C) 2003 Elsevier Ltd. All rights reserved.

Ultrastructure of the intercellular protuberances in leaves of Paepalanthus superbus Ruhl. (Eriocaulaceae)

Folhas adultas de Paepalanthus superbus mostram protuberâncias intercelulares, entre a parede periclinal interna das células epidérmicas e superfície de células parenquimáticas; depósitos semelhantes ocorrem na superfície das células parenquimáticas do mesofilo. Estas protuberâncias são mais proeminentes ao redor de células parenquimáticas, formando uma estrutura que lembra uma cápsula gelatinosa. Testes histoquímicos com vermelho de rutênio evidenciam sua natureza péctica, com inclusões lipídicas dispersas, detectadas por sudan IV e sudan black B. Ultra-estruturalmente as protuberâncias mostram matriz fibrilar permeada por estruturas fimbriadas e tubulares, com margem distinta formada por estrutura membranosa. Nossos resultados sugerem que estas protuberâncias são derivadas de atividade secretora, sendo formadas após o desenvolvimento dos espaços intercelulares. em P. superbus esta estrutura pode representar uma especialização da parede celular, relacionada com adesão e mecanismos de transporte entre células.

Cephalic salivary glands of two species of advanced eusocial bees (Hymenoptera: Apidae): morphology and secretion

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ultra-structure and histochemistry of digestive cells of Podisus nigrispinus (Hemiptera: Pentatomidae) fed with prey reared on bt-cotton

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Oil-soluble dyes for marking Spodoptera frugiperda (Lepidoptera : Noctuidae)

Although various biological aspects of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) have been examined, adult movement and dispersal of this insect pest is not well understood. Release-recapture techniques by using marked insects is a useful approach for dispersal studies; however, the marking technique should not significantly affect insect biology or behavior. Therefore, the effect of different concentrations of oil-soluble dyes (Solvent Blue 35 [C.I. 61554], Sudan Red 7B [C.I. 26050], Sudan Black B [26150], Sudan Orange G [C.I. 11920], and Sudan I 103624 [C.I. 12055]) on development, mortality, and fecundity of S. frugiperda was evaluated. Dyes were added to artificial diet used to feed larvae. Larval and pupal development and mortality, adult longevity, and female fecundity were evaluated. High concentrations (400 and 600 ppm) of all dyes led to longer larval and pupal stages. Adult life span and number of eggs were not affected by the dyes. Sudan Red 7B marked both adults and eggs very well. Solvent Blue 35 marked both adults and eggs, but the blue-marked eggs could not be distinguished from some bluish eggs laid by nonlabeled females. Adults and eggs were not adequately marked by the Sudan Black B, Sudan Orange G, and Sudan I 103624 (a yellow dye).

Características histoquímicas e estruturais do testículo do morcego hematófago (Desmodus rotundus rotundus, Geoffrey, 1810).

The testicular stroma of the vampire bat including the testicular capsula and the lamina propria of the seminiferous tubuli, was strongly PAS-positive. This observation was a possible indication of great amounts of structural glycogen and other glycoconjugates at the level of smooth muscle cells; elongated contractile cells and/or collagen frameworks of the tunica albuginea and tubular lamina propria. In the last the basement membranes of the seminiferous tubules were particularly strongly PAS positive, as an indication of their neutral mucosubstances structural composition, previously described (Malmi et al., 1987). The epithelium lining from the cavitary and surface rete testis complex showed low reactivities to mucosubstances; total proteins and lipids and oxidative enzymes studied. Although the apical granulation at the rete testis epithelium showed an intense PAS reactivity with hypothesis of glycoprotein secretion, through the rete. The PAS, Sudan Black B, NADH, MDH and LDH reactions of the testicular interstitium seem correlate to steroid metabolism (biosynthesis and secretion), at the Leydig cells level. The seminiferous epithelium generally had low reactions to all the histochemical studies realized. Particularly in the adbasal compartment the histochemical localizations of NADH diaphorase and LDH were possible related to glycolytic activities and general carbohydrates metabolism, both enzymes, and hydrogen transport, the NADH. The strong PAS, diastase and PAS, and alcian blue pH 2.5 and PAS reactions observed in the adluminal seminiferous epithelium compartment were directly related to the spermatids acrosomal glycoconjugates structuration. Also the SDH localization at this level seems to be related to the mitochondrial activities at the middle piece level in the late spermatids.

Testicular morphological changes during the seasonal reproductive cycle in the bullfrog Rana catesbeiana

Spermatogenesis and steroidogenesis undergo seasonal variations during the reproductive cycle in amphibians. Testicular morphological and morphometric seasonal variations as well as interstitial lipidic inclusions and intralobular glycoconjugates were evaluated during seasonal cycle of Rana catesbeiana. Testes of frogs collected during the annual seasons were weighed for calculation of GSI (Gonadosomatic index). Seminiferous lobule diameters (DSL) and volume densities of seminiferous lobules (VvSL), excretory ducts (VvED), and interstitial tissue (VvIT) were analyzed. Semithin sections were submitted to Periodic Acid-Schiff (PAS) and Alcian Blue (AB) methods for detection of glycoconjugates, while lipidic inclusions were detected by Sudan Black B. GSI showed no significant variations during the year. Since VvED and VvIT increased significantly during summer and were inversely proportional to VvSL, a compensatory effect between the testicular compartments may be related to the maintenance of GSI. During autumn/winter, larger lobular diameters were observed in comparison to spring/summer when spermiogenesis and spermiation were commonly observed. The increased VvIT and the numerous lipidic inclusions in the interstitial cells during summer suggest a relationship between spermiogenesis and steroidogenesis. Besides the structural stability variations occurring in the IT and SL, a possible paracrine interaction between ED and IT should be also involved in the IT development during summer. The presence of PAS and AB-positive globular structures were observed in the seminiferous lobules and excretory ducts. These structures containing acid glycoconjugates appear to be Sertoli cell apical portions, which are accumulated in the lumen of the seminiferous lobules mainly during spermiation. © 2004 Wiley-Liss, Inc.