Aldehyde-Fuchsin Followed by Toluidine Blue O for Pancreatic Islet Cells

A number of methods have been devised for the identification of the various cells of the pancreas. Currently, aldehyde-fuchsin (Gomori 1950) is one of the most extensively used stains for the β cells. To show other cells, Gomori suggested superimposing either an orange G or a trichrome stain. In our laboratory, we have been using a basic dye, following aldehyde-fuchsin for β cells, as a buffered solution of toluidine blue O (C.I. No. 52040) for $aL cells. This method is simple and practical, and yields satisfactory and reproducible results.     

Demonstration of neurosecretory substance in previously scanned specimens: adaptation of the Gomori aldehyde-fuchsin method for correlative SEM/TEM/LM histochemistry.

The Gomori aldehyde-fuchsin (AF) method for selective staining of neurosecretory substance (NSS) has been adapted to tissue previously prepared for both scanning and transmission electron microscopy (SEM/TEM). The procedure results in precise correlation of light microscopic (LM) histochemistry with SEM/TEM of the same tissue.     

Aldehyde-Thionin: A Stain Having Similar Properties to Aldehyde-Fuchsin

A mixture containing thionin, 0.5 gm; paraldehyde, 7.5 ml; concentrated HCl, 1 ml; and 70% ethanol, 91.5 ml, when allowed to ripen for several days, produces a stain which, when applied to sections of tissue fixed in a Zenker-based fixative, resembles in its effects the aldehyde-fuchsin stain of Gomori, but presents certain advantages.     

Quantitative estimation of islet tissue of pancreas in adult Grey kangaroos (Macropus fuliginosus)

Six Grey kangaroos, both male and female, were shot and weighed. The pancreas was isolated, weighed and samples fixed in Bouin's solution. Using a linear scanning technique (Carpenter & Lazarow, 1962) the mean islet tissue mass was estimated in head, neck, body and tail regions. A uniform distribution was found in all regions. The relationship between pancreas weight and body weight and islet mass in relation to body weight were calculated. Sections stained with aldehyde-fuchsin (Gomori, 1950), Mallory's phosphotungstic acid haemotoxylin and modified Davenport's technique (Epple, 1967) were prepared. Using a Wild-M501 semiautomatic sampling microscope and a Weibel graticule (Weibel et al., 1966) the percentage volume of β cells, α cells and δ cells were estimated respectively in the head, neck, body and tail regions of the six samples. The α cells or glucagon-producing cells were the most predominant as these macropodid marsupials are well adapted to dealing with hypoglycemic conditions. They were uniformly distributed in all regions.     

Influence of the feeding regimen on the rhythmicity of the processes of secretion of the supraoptic nucleus in C57Bl mice

During the period of vernal equinox in Leningrad 2 groups of C57Bl male mice have been investigated. Ninety-five animals are given food ecologically adequate at 9 p.m. Eighty-four animals are given foot at 9 a.m.--ecologically inverted regimen of feeding (IRF). The mice are decapitated for 4 days with an interval about 1.5 h. Serial paraffin sections are stained with aldehyde-fuchsin after Gomori and an additional staining of the nuclei with azocarmine. Criteria for the neurosecretory activity is the ratio of the cells amount at various stages of synthesis, outflux and accumulation of the secrete, volumes of the nuclei and nucleoli. Spectrum and parameters of the rhythmicity are revealed. IRF produces decrease in the amount of the ultradian component of the rhythmic parameters, characterizing active synthesis and discharge of the secrete. The part of the neurosecretory cells, those actively synthesizing and discharging the secrete, and volume of the cell nucleoli decrease. Range of ultradian component of the cell part rhythm, depositing the secrete, and the cell volume enriches. Thus, IRF produces certain changes in the rhythmicity of the cell secretion at all the stages: synthesis, discharge and accumulation of the secrete. Total intensity of the synthetic processes decreases. A conclusion is made that IRF inhibits the microsecretory process in the supraoptic nucleus (SON) and decreases adaptive possibilities of its cells. Adaptation to IRF is performed at the expense of rhythmic discharge of neurohormones, deposited in the cells, and at the expense of processes, occurring in the neuryoplasm and resulting in increase of the nuclear volume.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aldehyde-fuchsin: historical and chemical considerations.

The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections.     

Aldehyde-fuchsin: Historical and chemical considerations

Summary The staining mechanisms of Gomori's aldehyde-fuchsin are not yet fully understood. It seemed therefore timely to review the history of this dye class in context with current dye and aldehyde chemistry. In 1861 Lauth treated basic fuchsin with acetaldehyde. This dye became known as Aldehyde Blue, but consisted of violet and blue dyes. Schiff (1866) studied several aldehyde-fuchsins; these compounds contained two molecules of dye and three molecules of aldehyde. Acetaldehyde-fuchsin prepared according to Schiff's directions showed staining properties similar to those of Gomori's aldehyde-fuchsin. This dye class was soon superseded by new dyes more suitable for textile dyeing, and chemical investigations of aldehyde-fuchsins ceased around the turn of the century. Gomori's aldehyde-fuchsin has been regarded as a Schiff base. However, according to chemical data, low molecular aliphatic aldehydes and aromatic amines tend to form condensation products. Correlations of chemical and histochemical observations suggest such processes during aging of dye solutions. Models of dimers and polymers of aldehyde-fuchsin could be built without steric hindrance. The nature of the bonds formed by various components of aldehyde-fuchsin solutions is not clear. However, cystine in proteins, e.g. in basement membranes, apparently does not play a role in the binding of aldehyde-fuchsin by unoxidized Carnoy- or methacarn-fixed sections.     

Selective staining of hepatitis B surface antigen in thick epoxy sections of liver.

Hepatitis B surface antigen (HBsAg) in epoxy-embedded liver tissues can be stained by aldehyde-fuchsin stain. Sections are oxidized in KMnO4 acidified with H2SO4, then bleached in NaHSO3, both at 70 C. Heating for oxidation and bleaching are absolutely necessary. Diluted aldehyde-fuchsin stain adjusted to pH 1.5 to 1.8 with NaOH is used for staining. HBsAg is specifically stained purple. Other components such as mitochondria and bile pigments are also strained, but are easily distinguished from HBsAg. This staining method is advantageous for the identification of HBsAg-positive cells for electron microscopic observation.     

A Comparative Light Microscopic Evaluation of Oxytalan Fiber Staining with a Variety of Dye Substances

Staining of oxytalan fibers in marsupial, eutherian and human periodontal ligaments was surveyed with 65 different dyes. Using the criteria of responses to preoxidation, distribution, and morphologic appearance, 27 dye preparations in addition to the Gomori aldehyde-fuchsin Taenzera11 Uma orcein, and Weigert resorcin-fuchsin techniques displayed oxytalan fibers. With two exceptions all dyes were cationic and reacted with varying degrees of excellence with different animals. Most dyes produced their best staining results as concentrated solutions in 3% acetic acid, suggesting involvement of oxidatively engendered polyanions predominantly associated with an acid mucopolysaccharide component of the oxytalan fiber. The significance of carboxyl and sulfur-containing group should not be overlooked in further studies aiming to elucidate oxytalan fiber chemistry and microstructure. This study supported the view that oxytalan fibers belong to the family of elastic tissues and represent a biologically important system within the periodontal ligament.     

A comparative light microscopic evaluation of oxytalan fiber staining with a variety of dye substances.

Staining of oxytalan fibers in marsupial, eutherian and human periodontal ligaments was surveyed with 65 different dyes. Using the criteria of response to preoxidation, distribution, and morphologic appearance, 27 dye preparations in addition to the Gomori aldehyde-fuchsin, Taenzer-Unna orcein, and Weigert resorcin-fuchsin techniques displayed oxytalan fibers. With two exceptions all dyes were cationic and reacted with varying degrees of excellence with different animals. Most dyes produced their best staining results as concentrated solutions in 3% acetic acid, suggesting involvement of oxidatively engendered polyanions predominantly associated with an acid mucopolysaccharide component of the oxytalan fiber. The significance of carboxyl and sulfur-containing groups should not be overlooked in further studies aiming to elucidate oxytalan fiber chemistry and microstructure. This study supported the view that oxytalan fibers belong to the family of elastic tissues and represent a biologically important system within the periodontal ligament.     

3种淡水肉食性鱼类胰脏及胰液导管系统的组织学比较

应用解剖学和组织学技术对翘嘴鲌Culter alburnus、大鳍鳠Mystus macropterus和斑鳜Siniperca scherzeri的胰脏进行观察.结果表明,翘嘴鲌和大鳍鳠胰脏为弥漫混合型(suffusion-mix type),斑鳜为散布致密型(disperse-dense type).翘嘴鲌和大鳍鳠胰脏被膜较薄,厚度分别为4.33 μm±1.12 μm和7.24 μm±3.69 μm,而斑鳜胰脏被膜较厚,为50.96 μm±11.02 μm.翘嘴鲌胰小叶不明显,大鳍鳠非常明显,斑鳜比较明显.3种鱼胰腺细胞体积较大,核仁大而明显,细胞质内均有大量深色网络状或羽毛状结构和颗粒物质分布;胰脏内胰液运输管道均由闰管、小叶内导管、小叶间导管和集合导管组成,其中集合导管结构比较典型,管壁分为3层,即内层由单层柱状上皮和固有膜组成,中层由平滑肌细胞组成,外层由结缔组织组成.翘嘴鲌胰岛相对较小和分散,单位面积数量较少;大鳍鳠胰岛相对较大和分散,单位面积数量也较少;斑鳜胰岛最大,相对集中,单位面积数量较多.3种鱼胰岛外均被结缔组织被膜,结缔组织伸入胰岛内将胰岛细胞分隔为细胞小团或细胞索;G-醛复红染色仅观察到A细胞和B细胞,胞质颗粒分别被染成紫红色和黄色;B细胞占优势.     

Gimmicks of gamma‐aminobutyric acid (GABA) in pancreatic β‐cell regeneration through transdifferentiation of pancreatic α‐ to β‐cells

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma‐aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells’ (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA‐induced α‐cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α‐cell transformation to β cells in vivo and insulin‐secreting β‐like cells in vitro. On the other hand, GABA treatment showed that it has no α‐ to β‐cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA‐mediated transdifferentiation of α cells to β cells.     

Histochemical Localization of {beta}-Glycerophosphatase Activity in Young Root Tips

Glycerophosphatase activity has been studied in frozen sectionsof maize, barley, and broad-bean root tips by the Gomori leadsulphide precipitation procedure. The enzyme was largely localizedat particulate sites in the cytoplasm, and in the cell walls.In maize and barley the particles were similar to acid phosphatase-containingspherosomes found in other tissues and were most active in thecortical cells. The wall reaction was highest in the outer cells,in agreement with biochemical studies. When excised roots wereincubated in the Gomori medium, staining was restricted to thesurface cells. The possible function of this surface activityand its relevance to ultrastructural studies is discussed.     

Endocrine cells in the cardial glands of the esophagus in man

Distribution of endocrine cells in composition of the secretory epithelium of the cardial glands of the human esophagus in both sex and at various age has been investigated. In spiral paraffin slices the endocrine cells have been revealed by means of different silver impregnation methods (after Grimelius, Masson--Hamperl, Sevier--Munger), Sevke technique, ferry-ferrocyanide method. Some cells have been revealed, which according to the specific signs of their granule staining resemble very much G-, EC-, ECL-cells of the stomach. They can be triangular, flatten or polygonal and are stained in the cardial gland epithelium as single diffuse cells, or as groups of cells. Staining of the slices with aldehyde-fuchsin in various modifications reveals dark cells with dark-violet granules and lighter cells with acidophilic granules. Sometimes among these cells certain cells with light-violet cytoplasm are revealed. All these cells can be arranged both in composition of the secretory epithelium of the glands and in conglomerates of cells, resembling pancreatic islands. According to their tinctorial properties they resemble A-, B-, D-cells of these islands.     

The Human Insulin Receptor Cdna: A New Tool to study the Function of this Receptor

Abstract

The human insulin receptor (hIR) is an integral transmembrane glycoprotein comprised of two α and two β subunits. An immediate consequence of insulin binding to the extracellular α subunit is the autophosphorylation of tyrosine residues on the intracellular domain of the β subunit. The placental hIR cDNA has been cloned and sequenced, providing the primary structural features of the protein.

In order to investigate the functions of the β subunit and particularly the role of autophosphorylation and tyrosine phosphokinase (TPK) activity (a feature shared by other receptors and oncogene proteins) in transmembrane signalling, we designed an expression system of the hIR cDNA in eucaryotic cells. Superexpressing CHO cell lines that contain about 10⁶ functional hIR/cell have been developed. In these cells half maximum stimulation of glucose uptake occurs at 5x 10^-10M insulin, whereas normal CHO cells require 5x 10^-12M insulin. In this expression system we have carried out site-directed mutagenesis experiments in which domains of the molecule have been deleted or particular amino acids have been replaced by others. The replacement of either or both the tyrosine residues 1162 and 1163 compromise an autophosphorylated site that is important for kinase function and the insulin response. Expression of an isolated membrane-bound form of the β-subunit produces a 6 fold increase in glucose uptake. This insulin-independent effect disappears if the twin tyrosines are mutated or if the β subunit is expressed in the cytoplasm. These studies also show that the C terminal 112 amino acid portion of the β subunit is important for the stability of this protein.     

A quantitative method for measurement of lysosomal acid phosphatase latency in cultured rat heart cells with 210Pb

Synopsis A method is described for measuring the latency of lysosomal acid phosphatase in cultured rat heart endotheloid cells.²¹⁰Pb was added to a medium used to demonstrate acid phosphatase activity by the Gomori lead method, and the amount of lead deposited was measured with a liquid scintillation counter. Deposition rates were measured after enzyme activation pretreatments with acetate buffer (pH 5.0) at various osmolalities, and after formaldehyde fixation. Formaldehyde, alloxan, or fluoride in the Gomori medium were evaluated for their differential effects on lysosomal and non-lysosomal acid phosphatase. The method was found to provide a sensitive, rapid and quantitative evaluation of acid phosphatase latency and should be useful for studying the integrity of lysosomes within cells.     

Histochemical Evidence for the Presence of Lysosome-like Particles in root Meristem Cells of Vicia faba

The lysosomes of animal cells, as defined by biochemists, areparticles limited by a lipid-protein membrane and which containhydrolytic enzymes; they are inert until the permeability ofthe limiting membrane has been altered. In addition, the lysosomesin animal cells have been demonstrated also by histochemicalprocedures. Acid phosphatase may be used as a marker for lysosomes,and can be demonstrated by a modified Gomori procedure. Thus,in controlled-temperature frozen sections of plant tissue, ithas been possible to demonstrate granules which are inactivefor acid phosphatase until subjected to agents that will disruptlipid-protein structures, namely freezing and thawing, formaldehydeand heat. Whether such particles are the lysosomes is discussed.     

Monoamine-containing fibres in the pituitary neuro-intermediate lobe of the pig and rat

Summary A rich system of monoamine-containing fibres is described in the neural lobe and pars intermedia of the pig and rat. a) A rich network of delicate varicose fibres is evenly distributed throughout the parenchyma of the neural lobe and surrounds the cells of the pars intermedia. b) Droplets or clusters of droplets are scattered throughout the neural lobe. Most of them probably constitute terminal swellings or end-apparatuses of smooth or varicose fibres. The number of droplets varies from animal to animal; they are found also in the pars intermedia. c) Coarse varicose fibres are mainly localized around larger vessels. At least some of these fibres are nerve fibres of sympathetic origin. A combination of fluorescence microscopy and aldehyde-fuchsin staining on the same sections demonstrated that the majority at least of these monoamine-containing structures were not identical with aldehyde-fuchsin positive neurosecretory fibres.This research was supported by a grant from the Swedish Medical Research Council (B68-12X-712-03B) and by the Faculty of Medicine, University of Lund.     

Pancreatic β-cell regeneration: From molecular mechanisms to therapy

Pancreatic β cells are a type of cells that are present in the islets of Langerhans. These cells are highly specialized for the secretion of insulin in response to low increasing of blood glucose levels. Hence, pancreatic β cells could contribute to maintaining systemic glucose homeostasis. Increasing evidence has revealed that a variety of internal (ie, genetic and epigenetic factors) and external factors (ie, radical-oxidative stress) are involved in the protection and/or regeneration of pancreatic β cells. The pathways regulating β-cell replication have been intensely investigated. Glucose has an important role in cell cycle entry of quiescent β cells, which exerts its effect via glucose metabolism and unfolded proteins. A variety of growth factors, hormones, and signaling pathways (ie, calcium-calcineurin nuclear factor of activated T cells) are others factors that could affect β-cell replication under different conditions. Therefore, a greater understanding of the underlying pathways involved in the regeneration and protection of pancreatic β cells could lead to finding and developing new therapeutic approaches. Utilization of stem cells and various phytochemical agents have provided new aspects for preventing β-cell degeneration and stimulating the endogenous regeneration of islets. Thus, these therapeutic platforms could be used as potential therapies in the treatment of insulin-dependent diabetes mellitus. Here, we summarized the various mechanisms involved in pancreatic β-cell regeneration. Moreover, we highlighted different therapeutic approaches which could be used for the regeneration of pancreatic β cells.     

Electron microscopic localization of alkaline phosphatase in the trigeminal ganglion of the rat

Summary The electron microscopic demonstration of alkaline phosphatase (ALP) was carried out on the trigeminal ganglion of the rat using the calcium lead modification method by Gomori (Gomori, 1952; Molnar, 1952).The ALP reaction was localized on the junction of capsular cells and nerve cells, in the cytoplasm of some dark capsular cell and in that of the endothelial cell: The enzymatic reaction products (1) existed throughout the entire length of the junction of clear cells and capsular cells, (2) aggregated at some points of the junction of dark cells and capsular cells, (3) existed on the smooth and/or rough surfaced endoplasmic reticulum and on the ribosomes of some dark capsular cells.