Cytochemical Staining Method Research Articles

Cytochemical Detection of Peroxisomes in Light and Electron Microscopy with 3,3'-diaminobenzidine.

Peroxisomes are ubiquitous dynamic and multifunctional organelles that contribute to numerous anabolic and catabolic pathways, being essential for human health and development. Their best known functions include the oxidation of fatty acids and metabolism of hydrogen peroxide with catalase as a marker enzyme. Indeed, historically, it was the cytochemical staining of catalase in many different cells and tissues that revealed the ubiquitous presence of peroxisomes in almost all animal and plant cells. In this chapter, the method for cytochemical staining of catalase with the alkaline 3, 3'-diaminobenzidine (DAB) is described. Since aldehyde fixation is a prerequisite for staining of catalase with DAB, a method for perfusion fixation of rat liver with glutaraldehyde is presented prior to the cytochemical staining method and the subsequent tissue processing for light and electron microscopy.

BLT-esterase in infectious mononucleosis.

Peripheral blood lymphocytes of three patients suffering from infectious mononucleosis due to Epstein-Barr virus (EBV) infection were analysed for BLT-esterase expression in peripheral blood lymphocytes by a well established cytochemical staining method. During the acute phase of disease with presence of clinical symptoms a very high level of up to 90% BLT-esterase-expressing lymphocytes were detected. The increased percentage of lymphocytes expressing BLT-esterase coincided with the time of greatest symptoms and the peak elevation of hepatocellular enzymes. The still moderately elevated level only gradually decreased to normal during the further recovery period of 2 months during which the patients described episodes of weakness. Peripheral blood lymphocyte phenotype analysis revealed a marked CD8 lymphocytosis, a CD4/CD8 ratio of about 0.2, low number of CD19+ B cells, and a high level of DR+ CD3+ lymphocytes. Reduction of BLT esterase expression during the recovery period coincided with reduction of CD8+ DR+ lymphocytes. By a combination of BLT-esterase staining with immunocytochemical phenotype analysis, 95% of CD8+ lymphocytes were found to be BLT-esterase-positive. BLT-esterase might be involved in the immunodefence against EBV in infectious mononucleosis by inducing apoptosis in EBV-transformed B cells.

Localization of nucleolar DNA and transcription sites of rRNA genes <I>in situ in wheat</I>cells

By using the conventional electron microscopic technique and DNA specific cytochemical staining method (NAMA-Ur), we directly observed the arrangement and location of intranucleolar DNA in situ in wheat (Triticum aestivum L.) cells. The results showed that nucleolar DNA was found in Fibrillar Centers (FC), Dense Fibrillar Component (DFC) and the transitional region between FC and DFC. Moreover, the nucleolar DNA was distributed along the periphery of FC and by surrounding FC. We employed RNP preference staining (Bernhard staining) method to visualize the distribution and position of RNP in situ in nucleoli of wheat cells. The results directly showed that RNP mainly located in the transitional region between FC and DFC, in DFC and in Granular Component (GC). Moreover, RNP was irregularly distributed around FC. By employing anti-RNA/DNA hybrid antibodies, we directly and selectively labeled transcription sites of rRNA genes and testified that localization of transcription sites was not only in the transitional region between DFC and FC but also in DFC of nucleoli in wheat cells.

Biochemical and cytochemical evaluation of heterozygote individuals with glucose-6-phosphate dehydrogenase deficiency

The aim of this study was to diagnose heterozygous glucose-6-phosphate dehydrogenase (G6PD) deficient females by an inexpensive cytochemical G6PD staining method that is easy to perform, allowing diagnosis of G6PD deficiency without cumbersome genetic analysis. Three subject groups were included in the study. The first group consisted of 15 hemizygous deficient males. The second and the third group were composed of 15 heterozygous deficient females and 15 healthy individuals, respectively. Biochemical determination and cytochemical staining of G6PD activity were performed in samples of all subjects. Results obtained with the cytochemical staining method correlated significantly with the biochemical data (p < 0.001), but a only 51-68% of the erythrocytes were stained positively in females with normal biochemical G6PD activity despite their having a G6PD-deficient child. This observation clearly indicates that these individuals are heterozygously deficient. These findings show that the cytochemical staining method to detect G6PD activity in erythrocytes is reliable, sensitive and specific and is superior to the biochemical method. Therefore, this method can be used routinely to detect heterozygous G6PD deficiency.

Structural components of the nuclear body in nuclei of Allium cepa cells.

Nuclear bodies have long been noted in interphase nuclei of plant cells, but their structural component, origin and function are still unclear by now. The present work showed in onion cells the nuclear bodies appeared as a spherical structure about 0.3 to 0.8 microm in diameter. They possibly were formed in nucleolus and subsequently released, and entered into nucleoplasm. Observation through cytochemical staining method at the ultrastructural level confirmed that nuclear bodies consisted of ribonucleoproteins (RNPs) and silver-stainable proteins. Immunocytochemical results revealed that nuclear bodies contained no DNA and ribosomal gene transcription factor (UBF). Based on these data, we suggested that nuclear bodies are not related to the ribosome or other gene transcription activities, instead they may act as subnuclear structures for RNPs transport from nucleolus to cytoplasm, and may also be involved in splicing of pre-mRNAs.

Configuration of nucleolar DNAin situ in nucleolus ofAllium cepa cells

The location and configuration of nucleolar DNA have not been determined for a long time. In this paper, we have observed the nucleolar ultrastructure and the character of nucleolar DNA inAllium cepa cells by conventional electron microscopy and the cytochemical NAMA-Ur DNA specific staining method. Furthermore, we have properly improved the NAMA-Ur method so as to analyze the location and configuration of nucleolar DNAin situ. Our results indicated that the nucleolar DNA inAllium cepa cells is mainly located at the border between fibrillar centers and dense fibrillar component, especially distributed in the configuration of encircling the fibrillar centers.

Culture requirements and cytochemical characteristics of colony forming units-granulocyte/monocyte in cynomolgus monkeys

For the preclinical safety assessment of recombinant human haematopoietic growth factors, the in vitro bone marrow progenitor cell assays could provide valuable information that can be used to select appropriate biologically responsive animal species and to predict the potential toxicity and dosage selection for in-life toxicology studies. The cynomolgus monkey is a commonly used non-human primate model for the assessment of efficacy and toxicity of such compounds; however, the information regarding their bone marrow progenitor cell assays is scarce. This study evaluated the culture requirements and the morphological and cytochemical characteristics of colony forming units-granulocyte/monocyte (CFU-GM) for this species. The optimal CFU-GM growth was achieved in methylcellulose cultures of minimum essential medium-alpha (MEM-alpha) that contained 5% leucocyte-conditioned medium (LCM) and 30% heat inactivated horse serum (HIHS), after 14 days of incubation in a humidified atmosphere. A triple cytochemical staining method using naphthol-ASD chloroacetate esterase (CAE), alphanaphthol acetate esterase (ANAE) and luxol fast blue (LFB) was used to characterise the monocyte/macrophage, neutrophil and eosinophil colonies, respectively. This cytochemical method showed the presence of approximately 15% pure neutrophil, 61% pure monocyte/macrophage, 4% pure eosinophil, 15% mixed granulocyte/monocyte (positive for at least two stains) and 3.5% unstained colonies in 14-day-old cultures. The monocyte, neutrophil and mixed colonies were smaller in diameter, compact and contained a larger number of cells than the eosinophil colonies. Since eosinophil colonies exhibit lower cell density, they may be easily missed in total CFU counts performed at low magnification. These in vitro assays could be used to predict the potency of and lineage-specific response to haematopoietic growth factors in cynomolgus monkeys.

Cytochemical Demostration of Subtypes of Type I Cells in the Rabbit Carotid Body Chemoreceptor.

Samples of carotid bodies from control and reserpinized rabbits were incubated in a sodium chromate and potassium dichromate buffer after primary glutaraldehyde fixation. Such a cytochemical staining method could show that the reserpine treatment resulted in a decrease in the electron density of the densecored vesicles to a different degree in different subtypes of Type I cells. The decrease was moderate in subtype B cells, obvious in subtype C cells, and invisible in subtype A cells. The present work offered a morphofunctional evidence for the existence of subtypes of Type I cells, and gave a hint that the different subtype cells may store different catecholamines.

Morphometric evaluation of species differences in the effects of bezafibrate, a hypolipidemic agent, on hepatic peroxisomes and mitochondria.

In order to demonstrate the species differences in the effects of a hypolipidemic agent, bezafibrate, on hepatic peroxisomes and mitochondria, bezafibrate a hypolipidemic agent was administered orally to rats, mice, guinea pigs, rabbits, and dogs for two weeks, and to monkeys for 13 weeks. Peroxisomes in the livers were visualized using a cytochemical staining method for catalase with diaminobenzidine. Morphometric comparison of the hepatic peroxisomes and mitochondria was made to examine by point counting method. Peroxisome proliferation was the highest among male rats (4.15 fold) and then male and female mice (3.68 fold and 4.47 fold respectively), with a more moderate increase of this organelle in guinea pigs and monkeys, whereas no change was seen in rabbits and dogs. A slight increase in the volume density of mitochondria was observed for female rats, guinea pigs, and male and female monkeys. These results show that there are marked species differences in the effects of bezafibrate on hepatic peroxisomes and mitochondria. We considered that the species differences are very important to elucidate the mechanism of peroxisomal proliferation, hypolipidemic action, carcinogenicity, and their relationship.

Effect of fixation on activity and cytochemistry of hydrogenosomal enzymes in Trichomonas vaginalis

The effect of fixation on the activity of malate dehydrogenase (decarboxylating) and pyruvate synthase was investigated in Trichomonas vaginalis. Subsequently a cytochemical staining method was developed for the demonstration of malate dehydrogenase activity in hydrogenosomes. After fixation of cells in low concentrations of glutaraldehyde and incubation in the presence of malate and the tetrazolium compound 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. During the whole procedure strictly anaerobic conditions were required. Attempts to develop an analogous procedure for pyruvate synthase failed because even low concentrations of glutaraldehyde strongly inhibited enzyme activity. When cells were fixed in low concentrations of glycolaldehyde and acetaldehyde, a high enzyme activity was retained, but no staining could be achieved. Application of both staining methods to the sapropelic ciliates Trimyema compressum and Plagiopyla nasuta gave negative results.

Upregulation of a lymphoid serine protease in kidney allograft recipients

The presence of a putative, cytotoxicity-linked lymphoid serine esterase (SE) has been studied in 79 kidney graft recipients. Peripheral blood lymphocytes (PBL) bearing an N-alpha-benzyloxy carbonyl-L-lysine thiobenzyl ester (BLT)-specific SE were evaluated by a novel cytochemical staining method. A characteristic of post-allograft patients was an increased presence of SE containing granules in PBL. In 46 patients with stable graft function SE + PBL were 33.41 +/- 10.34% (controls: 26.30 +/- 5.22%, P less than 0.0025), SE + CD4+ 4.32 +/- 3.85% (controls 2.13 +/- 1.52%, P less than 0.0025) and SE + CD8+ T cells 47.68 +/- 18.64% (controls: 28.50 +/- 6.50%, P less than 0.0005). In those graft recipients undergoing a rejection episode a marked upregulation of SE activity could be observed when compared to the stable graft group: SE + PBL were 59.91 +/- 10.89% (P less than 0.0005), SE + CD8+ 74.30 +/- 10.79% (P less than 0.0005) and SE + CD4+ T cells 28.56 +/- 13.50% (P less than 0.0005). In 10 cases this increase of SE activity was observed with a time lag of up to 37 days prior to the onset of clinical or biopsy proven rejections, promptly decreasing in response to methylprednisolone antirejection therapy. In patients with recurrent rejection episodes and subsequent graft loss, a repeating increase of SE activity indicated a failure of therapeutic agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison between the chromate inhibition test and a cytochemical method for the determination of glucose-6-phosphate dehydrogenase deficiency in erythrocytes

The sensitivity and specificity of the chromate inhibition test for the determination of glucose-6-phosphate dehydrogenase (G6PD) deficiency in erythrocytes were compared with a cytochemical staining method. Fifty blood samples were used in a double blind study. The samples were selected from 600 blood samples on the basis of two biochemical criteria, viz. either G6PD activity < 4.8 IU/g Hb as analysed spectrophotometrically and/or G6PD activity less than glutathione reductase (GSSG-R) activity. The cytochemical assay was taken as reference because it has been proved to be sensitive and specific for the detection of heterozygous and homo hemizygous forms of deficiency. Cytochemically, one hemizygously deficient patient, 19 heterozygotes and 30 normals were detected. When applying the chromate inhibition test a somewhat different result was obtained with the same samples: one of the 30 normals was classified as heterozygously deficient (3% false positives) and 5 of the 19 heterozygously deficient patients were classified as normal (26% false negatives). It is concluded that the chromate inhibition test is a more sensitive biochemical test than the fluorescence spot test or spectrophotometric assays. However, it is less reliable than the cytochemical test for the detection of heterozygously G6PD deficient patients.

Zona Pellucida-Induced Acrosome Reaction in Boar Sperm1

Induction of the acrosome reaction in boar sperm by the zona pellucida (ZP) was investigated. A modified cytochemical staining method for measuring the acrosome reaction in boar sperm gave equivalent results to those obtained with transmission electron microscopy. Isolated heat-solubilized ZP effectively induced the acrosome reaction in boar sperm at a concentration of 25 micrograms/ml. Electrophoretically purified ZP components were also tested for acrosome reaction-inducing activity; both the 55,000 and 90,000 components of the ZP were effective. The carbohydrate moiety of the 55,000 component was necessary for activity because the polypeptides derived by chemical deglycosylation of the two glycoproteins did not induce the acrosome reaction.

Cytochemical Localization of Hydrogenase Activity in the Anaerobic Protozoa Trichomonas vaginalis, Plagiopyla nasuta and Trimyema compressum

A cytochemical staining method was developed for the demonstration of hydrogenase activity in hydrogenosomes of Trichomonas vaginalis. After fixation in low concentrations of glutaralde-hyde under strictly anaerobic conditions and incubation of these cells in the presence of H2 and the tetrazolium compound 2-(2′-benzothiazolyl)-5-styryl-3-(4′-phthalhydrazidyl)-tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. The method was used to demonstrate the hydrogenosomal nature of similar microbodies of the sapropelic ciliates Plagiopyla nasuta and Trimyema compressum.

Myeloperoxidase deficient polymorphonuclear leucocytes: computerized planimetric estimations of cellular and nuclear size.

Quantitative estimations of the mean areas of cell, nucleus and cytoplasm in polymorphonuclear leucocytes (PMN) were performed by automated image analysis of blood smears from six patients with acute myeloid leukaemia. The PMN were qualitatively separated by a cytochemical staining method into two well-defined subpopulations i.e. myeloperoxidase (MPO)-normal and MPO-deficient PMN. MPO-deficient PMN were characterized by a decreased size of the total cell (P less than 0.01), an increased size of the nucleus (P less than 0.01) and a decreased size of the cytoplasm (P less than 0.01). The resulting highly increased nucleus-to-cytoplasm ratio in this specific PMN subpopulation bears a striking resemblance to cells in malignant tumours. The planimetric results in this study further support the concept that MPO-deficient PMN may be the progeny of leukaemic precursors.

A SENSITIVE CYTOCHEMICAL STAINING METHOD FOR IN INDIVIDUAL ERYTHROCYTES GLUCOSE‐6‐PHOSPHATE DEHYDROGENASE (G6PD) ACTIVITY

British Journal of HaematologyVolume 63, Issue 2 p. 400-402 Free Access A SENSITIVE CYTOCHEMICAL STAINING METHOD FOR IN INDIVIDUAL ERYTHROCYTES GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) ACTIVITY JOAN LLUIS VIVES Corrons, JOAN LLUIS VIVES Corrons Servei de Laboratorie d'Hematologia and Escola Professional d'Hematologia‘Farreras Valentf, Hospital Clinic i Provincial, c I Casanova, 143-08036 Barcelona, SpainSearch for more papers by this authorAnna Merino, Anna Merino Servei de Laboratorie d'Hematologia and Escola Professional d'Hematologia‘Farreras Valentf, Hospital Clinic i Provincial, c I Casanova, 143-08036 Barcelona, SpainSearch for more papers by this authorMARIA ASSUMPCIO Pujades, MARIA ASSUMPCIO Pujades Servei de Laboratorie d'Hematologia and Escola Professional d'Hematologia‘Farreras Valentf, Hospital Clinic i Provincial, c I Casanova, 143-08036 Barcelona, SpainSearch for more papers by this author JOAN LLUIS VIVES Corrons, JOAN LLUIS VIVES Corrons Servei de Laboratorie d'Hematologia and Escola Professional d'Hematologia‘Farreras Valentf, Hospital Clinic i Provincial, c I Casanova, 143-08036 Barcelona, SpainSearch for more papers by this authorAnna Merino, Anna Merino Servei de Laboratorie d'Hematologia and Escola Professional d'Hematologia‘Farreras Valentf, Hospital Clinic i Provincial, c I Casanova, 143-08036 Barcelona, SpainSearch for more papers by this authorMARIA ASSUMPCIO Pujades, MARIA ASSUMPCIO Pujades Servei de Laboratorie d'Hematologia and Escola Professional d'Hematologia‘Farreras Valentf, Hospital Clinic i Provincial, c I Casanova, 143-08036 Barcelona, SpainSearch for more papers by this author First published: June 1986 https://doi.org/10.1111/j.1365-2141.1986.tb05569.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume63, Issue2June 1986Pages 400-402 ReferencesRelatedInformation

Regulatory peptides in Barrett's oesophagus.

Barrett's epithelium refers to the presence of ectopic mucosal types in the squamous-lined oesophagus. Previous studies have documented argentaffin and argyrophil-positive cells as well as gastrin-like immunoreactivity in oesophageal tissue extracts from patients with Barrett's mucosa. In the present study, 125 oesophageal biopsies obtained under direct vision at endoscopy from 22 patients with Barrett's oesophagus were systematically studied using fluorescence and peroxidase antiperoxidase single and double-staining immunocytochemical methods employing highly specific antibodies to localize the following peptide-containing cell types in Barrett's mucosa: gastrin, somatostatin, gastric inhibitory polypeptide, motilin, neurotensin and pancreatic glucagon. In addition, EC cells were localized using a cytochemical silver staining method. The results of this study indicate that EC cells and gastrin- and somatostatin-containing endocrine cells are detectable in Barrett's epithelium.

A sensitive cytochemical staining method for glucose‐6‐phosphate dehydrogenase activity in individual erythrocytes II. FURTHER IMPROVEMENTS OF THE STAINING PROCEDURE AND SOME OBSERVATIONS WITH GLUCOSE‐6‐PHOSPHATE DEHYDROGENASE DEFICIENCY

A cytochemical method for staining glucose-6-phosphate dehydrogenase (G6PD) activity in individual erythrocytes as reported previously has been optimized further by the incorporation of a number of technical improvements. Analysis of the enzyme content in erythrocytes of normal individuals as well as patients suffering from G6PD deficiency in the homozygous and heterozygous forms allows these three categories to be easily distinguished. Considerable formazan production occurs in most erythrocytes of a healthy person and only a small percentage of the cells appeared to be negative. Two cell populations of almost equal size could be discerned in heterozygotes for G6PD deficiency, one completely negative, the other with a variable amount of formazan per cell. Homozygous deficiency leads to a population of negative cells with a few positive ones after staining. It is concluded that a reliable method has been found for analysis of G6PD deficiency in erythrocytes at the single cell level.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. I. Optimalization of the staining procedure.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also for quantitative localization of its activity in individual erythrocytes. The staining procedure in its optimal form consists of a treatment of the erythrocytes with sodium nitrite, then a "fixation" in 0.025% glutaraldehyde (under NADP+ protection of the active site of the enzyme), followed by incubation of the cells in suspension in the presence of tetranitro BT, 1-methoxyphenazine methosulphate and polyvinyl alcohol. Using this new technique, a sharp localization is obtained of the glucose-6-phosphate dehydrogenase activity, which enables discrimination between red cells with different levels of enzyme activity, as a consequence of enzyme deficiencies or age changes.

Acid phosphatase activity in secretory granules of pancreatic beta cells of normal rats.

A cytochemical staining method for acid phosphatase combined with electron microscopy was applied to the islets of Langerhans of adult normal rats. In addition to dense bodies, some Golgi cisternae as well as a varying number of secretory granules of β cells were found to contain acid phosphatase. In view of the paucity of the knowledge concerning the role of acid phosphatase, the possible functional significance of its presence in the secretory granules of pancreatic β cells cannot be denned at the present time.