Basic and 'special' stains for plastic sections in bone marrow histopathology, with special reference to May-Grünwald Giemsa and enzyme histochemistry

A battery of morphological, histochemical, and enzyme histochemical stains have been experimented on semithin sections of glycol-methacrylate-embedded bone marrow biopsies. We have been able to reproduce on sections the typical 'Romanowsky effect' which characterizes May-Grünwald Giemsa-stained smears of bone marrow or peripheral blood. This appears to be of critical importance for proper routine morphological evaluation of bone marrow biopsies. Conventional histochemical stains, and the enzyme histochemistry reactions that are most useful and widely used in the study of marrow aspiration smears have been successfully applied to plastic sections: in this way the evaluation of the cytochemical profiles of marrow diseases, especially leukemias, may be included in the histopathologist's diagnostic approach, with the additional advantage of preserving the architecture of the tissue and the relationship between haemapoietic cells and stromal components.     

Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture.

A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity. Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed. In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP). We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.     

Histochemistry of carbohydrates as performed by physical development procedures

Summary The histochemistry of carbohydrates demonstrated by means of physical development procedures has been reviewed in terms of the use and reliability of the procedures, physical developers, practice of the procedures, a fundamental series of light and electron microscopic methods and certain other promising aspects of this area of histochemistry. A line of fundamental light- and electron-microscopic histochemical methods for carbohydrates using physical development procedures such as periodic acid thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD), high- or low-iron diamine (HID or LID)-TCH-SP-PD and lectin-gold (LT-G)-PD and related methods has been found to be more efficient, compared with those without physical development procedures. Since a series of other promising histochemical methods for carbohydrates using physical development procedures have been derived or are now being introduced, these procedures could be regarded as an unusually potent vehicle for effectively advancing carbohydrate histochemistry in both light and electron microscopy.     

Histochemistry of flight muscles in the Jamaican fruit bat, Artibeus jamaicensis: implications for motor control

Two fast-twitch fiber types are histochemically identified in the primary flight muscles of Artibeus jamaicensis. These are classified as type IIa and IIb according to an acid-preincubation staining protocol for myosin ATPase. All fibers in the bat flight muscles exhibit relatively intense staining properties for NADH-TR, suggesting a high oxidative capacity. The glycolytic potential of all fibers is rather low, as assessed by stains for alpha-GPD. This two-type histochemical profile appears to parallel biphasic electromyographic patterns observed in these muscles and leads us to propose that flight muscle histochemistry and activation are mediated by a "two-gear" neuromuscular control system. In contrast, earlier studies on Tadarida brasiliensis demonstrate the existence of a "one-gear" neuromuscular control system, exemplified by the presence of one fiber type. These observations are discussed with respect to the natural history and flight styles of several species.     

The tetrazolium-formazan system: design and histochemistry.

Our increasing knowledge about the chemistry and the correlations between chemical structure and histochemical properties of the tetrazolium/formazan system is resulting in: a better understanding of existing histochemical tetrazolium techniques; the selection of optimal tetrazolium salts for qualified use in histochemistry, cytochemistry and biochemistry; both qualitative and quantitative improvements in histochemical techniques for purposes demonstrating the activities of various dehydrogenating enzymes; an extended insight into the "state" of the tested biological object by means of tetrazolium indicators with special properties; and the combination of histochemical enzyme determination with further morphological techniques. This article has attempted to illustrate the progress in the use of the tetrazolium/formazan-system for histochemical purposes.     

Aspiration biopsy cytology and special stains

Histochemical methods play a vital role in the histology laboratory. The application of histochemistry to aspiration biopsy cytology (ABC) has shown equal promise in the demonstration of distinctive intracytoplasmic and extracytoplasmic substances. In our examination of more than 8,000 fine needle aspirates, we have found that certain histochemical studies may be of special diagnostic importance. These include mucicarmine, periodic acid-Schiff, oil red O, Fontana-Masson argentaffin and Gomori's iron stains. With the exception of the oil red O stain for neutral lipids (alcohol soluble), each may be applied to the destained smear initially prepared according to the method of Papanicolaou. Modifications of these histologic stains have been made to suit the "rapid service" motto of our busy cytology laboratory.     

Identification of 5-Bromo-2’-Deoxyuridine-Labeled Cells during Mouse Spermatogenesis by Heat-Induced Antigen Retrieval in Lectin Staining and Immunohistochemistry

DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2’-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes.     

On the future contents of a small journal of histochemistry

In the last three years, more than 70,000 scientific articles have been published in peer reviewed journals on the application of histochemistry in the biomedical field: most of them did not appear in strictly histochemical journals, but in others dealing with cell and molecular biology, medicine or biotechnology. This proves that histochemistry is still an active and innovative discipline with relevance in basic and applied biological research, but also demonstrates that especially the small histochemical journals should likely reconsider their scopes and strategies to preserve their authorship. A review of the last three years volumes of the European Journal of Histochemistry, taken as an example of a long-time established small journal, confirmed that the published articles were widely heterogeneous in their topics and experimental models, as in this journal''s tradition. This strongly suggests that a journal of histochemistry should keep its role as a forum open to an audience as broad as possible, publishing papers on cell and tissue biology in a wide variety of models. This will improve knowledge of the basic mechanisms of development and differentiation, while helping to increase the number of potential authors since scientists who generally do not use histochemistry in their research will find hints for the applications of histochemical techniques to novel still unexplored subjects.Key words: Basic and applied histochemistry     

Bioautography: A general method for the visualization of isozymes

A new method has been developed for visualization of isozymes which are difficult or impossible to detect with standard histochemical or autoradiographic methods. The principle of this method, bioautography, is the use of a microbial reagent to locate an enzyme after gel electrophoresis. When bioautography was compared to other staining procedures, the bioautographic method yielded identical results to those observed by the histochemical method for lactate dehydrogenase (LDH) or by the autoradiographic method for the adenine phosphoribosyltransferase (APRT). Using the bioautographic method, stains for enzymes which could not be visualized by any other procedure have been developed: argininosuccinate lyase and branched-chain aminotransferase. By employing appropriately genetically marked bacterial strains, it should be possible to develop new isozyme stains for a large number of unstudied isozymes.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Robust automated tumour segmentation on histological and immunohistochemical tissue images

Tissue microarray (TMA) is a high throughput analysis tool to identify new diagnostic and prognostic markers in human cancers. However, standard automated method in tumour detection on both routine histochemical and immunohistochemistry (IHC) images is under developed. This paper presents a robust automated tumour cell segmentation model which can be applied to both routine histochemical tissue slides and IHC slides and deal with finer pixel-based segmentation in comparison with blob or area based segmentation by existing approaches. The presented technique greatly improves the process of TMA construction and plays an important role in automated IHC quantification in biomarker analysis where excluding stroma areas is critical. With the finest pixel-based evaluation (instead of area-based or object-based), the experimental results show that the proposed method is able to achieve 80% accuracy and 78% accuracy in two different types of pathological virtual slides, i.e., routine histochemical H&E and IHC images, respectively. The presented technique greatly reduces labor-intensive workloads for pathologists and highly speeds up the process of TMA construction and provides a possibility for fully automated IHC quantification.     

Fasciola hepatica: ultrastructure and histochemistry of the glycocalyx of the tegument.

The morphology and histochemistry of the glycocalyx of the tegument was investigated by electron microscopy. The results showed that both the morphology and histochemistry of the glycocalyx varied depending on the environment immediately prior to fixation and also on postfixation treatments. Conventional electron microscope fixation appeared to preserve only about half the total thickness of the glycocalyx, and only histochemical tests applied en bloc gave a true morphological and histochemical picture. The glycocalyx, therefore, consists of two parts, an inner continuous layer which is tightly bound to the apical plasma membrane and is always preserved, and an outer fibrillar layer, which is not always preserved by conventional fixation. Both layers are anionic and carbohydrate-rich and therefore contain glycoproteins and sialic acids. The surface of Fasciola hepatica, therefore, has morphological and chemical features very like those proposed for the “greater membrane” by Lehninger.     

Simultaneous visualization of immunodetected antigens and tissue components revealed by non-enzymatic histochemical stains.

We explored the possibility of simultaneous application of histochemical and immunohistochemical staining techniques on the same paraffin-embedded human tissue section. Conventional histological stains (PAS, Alcian, Alcian-PAS, Van Gieson, Gomori silver impregnation, and Giemsa) were used in association with a battery of markers (keratins, leucocyte common antigen, S-100 protein, Factor VIII-related antigen) that are widely employed in diagnostic and experimental studies. We found that the best procedure was to perform immunostaining before the histochemical reaction, as this enables all the other possible combinations to be carried out. In addition, several detection systems, such as peroxidase-anti-peroxidase (PAP), alkaline phosphatase-anti-alkaline phosphatase (APAAP), and avidin-biotin complex (ABC), were tested and all gave consistent results. Some minor modifications of the histological staining methods were necessary, but the current immunohistochemical techniques could be used as established. Preliminary findings indicate that immunohistochemistry can be combined with histochemistry techniques by means of a relatively simple procedure whose only disadvantage is the time required to carry out the double staining.     

Unravelling the Philosophies Underlying ‘Animal Personality’ Studies: A Brief Re‐Appraisal of the Field

The last decade has seen lots of studies on ‘animal personality’ (i.e. the study of consistent between‐individual behavioural differences). As timely and promising as this field is, its development has come with a diversity of research questions. As an unfortunate consequence, it now suffers from substantial confusion about what ‘animal personality’ is, and how relevant related research frameworks are. Here, we stress the current inconsistencies and sources of confusion pertaining to the field, and their consequences on terminology used and miscommunication between researchers. In an attempt to unravel and clarify the concepts underlying the field, we identify two distinct, but complementary, theory‐driven conceptual frameworks: the intra‐individual variability (IIV) approach and the life‐history (LH) approach, which we believe encompass the vast majority of existing ‘personality studies’. Finally, we argue in favour of theory‐driven studies of consistent behavioural differences and state that the integrative statistical properties of random regression models should not override the merit of alternative conceptual frameworks. We then provide brief guidelines and warnings for a parsimonious and sound use of terminology.     

A new polychrome stain and simultaneous methods of histological, histochemical and immunohistochemical stainings performed on semithin sections of Bioacryl-embedded human tissues

Summary We describe a new polychrome stain and simultaneous methods of histological, histochemical and immunocytochemical staining performed on sections from human tissues embedded in the new hydrophilic resin Bioacryl. The polychrome stain involves the sequential use of Harris' Haematoxylin, silver methenamine, Light Green and Eosin or Safranin dyes and provides a highly specific visualization of the overall cytological tissue architecture. When histochemical, immunocytochemical, and polychrome stains are performed together on the same section, crisp images are obtained, yielding simultaneous data of histochemical and immunological reactivities with clear tissue architecture.     

Immunohistochemical/Histochemical Double Staining Method in the Study of the Columnar Metaplasia of the Oesophagus

Intestinal metaplasia in Barrett’s oesophagus (BO) represents an important risk factor for oesophageal adenocarcinoma. Instead, few and controversial data are reported about the progression risk of columnar-lined oesophagus without intestinal metaplasia (CLO), posing an issue about its clinical management. The aim was to evaluate if some immunophenotypic changes were present in CLO independently of the presence of the goblet cells. We studied a series of oesophageal biopsies from patients with endoscopic finding of columnar metaplasia, by performing some immunohistochemical stainings (CK7, p53, AuroraA) combined with histochemistry (Alcian-blue and Alcian/PAS), with the aim of simultaneously assess the histochemical features in cells that shows an aberrant expression of such antigens. We evidenced a cytoplasmic expression of CK7 and a nuclear expression of Aurora A and p53, both in goblet cells of BO and in non-goblet cells of CLO, some of which showing mild dysplasia. These findings suggest that some immunophenotypic changes are present in CLO and they can precede the appearance of the goblet cells or can be present independently of them, confirming the conception of BO as the condition characterized by any extention of columnar epithelium. This is the first study in which a combined immunohistochemical/histochemical method has been applied to Barrett pathology.Key words: Barrett’s oesophagus, columnar-lined oesophagus, p53, Aurora A kinase     

The history,chemistry and modes of action of carmine and related dyes

Carmine has been used in biological staining to demonstrate selectively nuclei, chromosomes or mucins, depending on the formulation. Throughout its history in science, complaints and frustrations have been expressed about dye quality. Inconsistencies in dye quality or identity have prevented thorough understanding of staining mechanisms and have caused many stain solutions to behave unsatisfactorily. The aim of this review is to (1) detail causes of these problems, which are rooted in history, geography and production, (2) offer ways to minimize problems and (3) provide modern explanations for stain behavior. Carmine is a “semi-synthetic” dye, i.e., a complex of aluminum and the natural dye cochineal (carminic acid). Carmine shows considerable batch-to-batch variability. Geography, politics, history, agricultural practices and iconography all contribute to the variability of cochineal. In addition, widely divergent manufacturing methods are used to produce carmine. Also, confusion in terminology has led to mislabeling. Pressure from the food industry for a more satisfactory colorant for acidic foods led to the introduction of a new dye, aminocarminic acid, which could enter the biological market inadvertantly. Improved methods of analysis should help the certification process by the Biological Stain Commission. Further standardization could be achieved by replacing most of the methods of solubilizing carmine. The majority of these methods use heat, which is likely to damage the dye molecule. Fortunately, carmine is readily dissolved by raising the pH of the aqueous solvent above 12, and a new form of the dye, now available commercially, is soluble in water without the need for heat or pH adjustment. Chemical structures and physical properties of carminic acid, carmine, aminocarminic acid and kermesic acid are reviewed. A new configuration for carmine is proposed, as well as possible changes to carminic acid and carmine molecules as a result of decomposition caused by heating. Each of the major classes of carmine-based stains is described as are possible mechanisms of attachment to specific substrates. Glycogen binds carmine through hydrogen bonding, and it is here that carmine decomposed by heat could have the greatest detrimental impact. Nuclei and chromosomes are stained via coordination bonds, perhaps supplemented by hydrogen bonds. Finally, acidic mucins react ionically with carmine. Specificity in the latter case may be due to unique polymeric carmine molecules that form in the presence of aluminum chloride.     

Newer applications of the histological stain prepared from Pterocarpus santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.