Bioautographic visualization of dihydrofolate reductase in enzyme overproducing BHK mutants

A bioautographic procedure has been developed for the visualization of the isozymes of dihydrofolate reductase (DHFR, E.C. 1.5.1.3). In addition to detecting electrophoretically separated enzymes, bioautography was utilized to visualize DHFR after isoelectric focusing on polyacrylamide gels. Both zone electrophoresis and isoelectric focusing were used to compare wildtype BHK cells to mutants which overproduce dihydrofolate reductase. In agreement with other physical data, the BHK-A5 overproduction mutant appears to produce more dihydrofolate reductase of the same electrophoretic mobility and isoelectric point as wild type cells.This study was supported by Grants GM 21433 and CA 19019 from the National Institutes of Health.     

Two-dimensional spectroscopy of electrophoretic gels

Optical techniques are described which permit one to analyze two-dimensional electrophoretic gels in a fashion which is analogous to the one-dimensional spectroscopy of solutions. In the methods described, an electrophoretic gel is irradiated with monochromatic light and isozyme patterns are detected by the absorption of light or the fluorescent emission of light. The system described can both generate and detect monochromatic light in a range from 200 to 1100 nm. Without the use of histochemical stains, several isozymes have been visualized by purely optical means. Five methods for the visualization of lactate dehydrogenase and five methods for the demonstration of trypsin isozymes are described. In addition, general methods have been formulated for hydrolases and oxidases. Gel spectroscopy should permit the investigation of a wide range of new isozymes.This work was supported in part by NIH Grants CA 19017 and GM 21433.     

Isozymal differention between two species of Prosopis

The patterns of five multilocus isozyme systems were investigated in seed, shoot and cotyledon tissue of two species of mesquite, Prosopis glandulosa var. glandulosa and P. pallida. The isozymes of malate dehydrogenase, peroxidase, esterase, alcohol dehydrogenase and acid phosphatase from each of these tissues were analysed by starch gel electrophoresis and specific histochemical stains. In the case of each enzyme system examined, there were distinctly different isozymes which could be utilized to differentiate between these two species.     

Bisbenzimide: a fluorescent counterstain for tissue autoradiography

Interpretation of the data from experiments using autoradiography (e.g. using in situ hybridization histochemistry, receptor binding, neuronal tract-tracing etc.) is aided when the autoradiographic grains can be seen in the context of cellular boundaries. Studies making use of autoradiography in the central nervous system have sometimes used tinctorial stains, such as cresyl violet, as counterstains to visualize the labeling. Tinctorial stains are excellent Nissl stains however, under bright-field illumination such dyes tend to obscure autoradiographic grains. In addition, dark-field illumination provides a common means of visualizing autoradiographic grains but tictorial stains are not optimally visible under these conditions. In an effort to find a counterstain that would be compatible with dark-field illumination, we have investigated the use of fluorescent dyes. Of the fluorescent dyes tested, bisbenzimide (Hoechst 33258) in pH 2.0 buffer was found to be optimal. Bisbenzimide counterstaining gave good resolution of cellular boundaries and appeared not to interfere with the ability to visualize autoradiographic grains. Furthermore, the illumination of bisbenzimide and of the autoradiographic grains could be controlled independently, making it easy to visualize or photograph the bisbenzimide Nissl staining and the autoradiographic grains simultaneously. Thus bisbenzimide is well suited for use as a fluorescent counterstain in autoradiographic studies.     

Automatic quantitative evaluation of autoradiographic band films by computerized image analysis

The present paper describes a new image processing method for automatic quantitative analysis of autoradiographic band films. It was developed in a specific image analysis environment (IBAS 2.0), but the algorithms and methods can be utilized elsewhere. The program is easy to use and presents some particularly useful features for evaluation of autoradiographic band films, such as the choice of whole film or single lane background determination; the possibility of evaluating bands with film scratch artifacts and the quantification in absolute terms or relative to reference values. The method was tested by comparison with laser-scanner densitometric quantifications of the same autoradiograms. The results show the full compatibility of the two methods and demonstrate the reliability and sensitivity of image analysis. The method can be used not only to evaluate autoradiographic band films, but to analyze any type of signal bands on other materials (e.g. electrophoresis gel, chromatographic paper, etc.).     

A C. elegans mutant screen based on antibody or histochemical staining

A method has been developed for isolating mutations in Caenorhabditis elegans that alter antibody or histochemical staining patterns. The basis for this method is a new procedure for making C. elegans permeable that does not kill the eggs contained within the uterus of gravid adult hermaphrodites. A mutagenized population of gravid hermaphrodites is made permeable and then stained with either an antibody or a histochemical stain. Animals that stain aberrantly are picked to individual petri plates and the eggs within the uterus of the stained mother hatch and establish a new genetic line. Antibody and histochemical stains are especially useful phenotypes because the staining pattern will usually directly reflect the gene expression pattern of the gene that codes for the antigen or enzyme. This method was used to isolate mutants that alter the expression of a mec-7lacZ fusion gene. Transgenic animals that contained the mec-7lacZ gene integrated into chromosome I were treated with the mutagen ethylmethanesulfonate, allowed to self-fertilize for two generations and then stained with X-gal or antibodies against β-galactosidase. Gravid animals that stained abnormally were picked to fresh petri plates and offspring were used to establish new mutant lines.     

Ruthenium hexaammine trichloride chemography for aggrecan mapping in cartilage is a sensitive indicator of matrix degradation.

We developed a new quantitative histochemical method for mapping aggrecan content in articular cartilage and applied it to models of cartilage degradation. Ruthenium hexaammine trichloride (RHT) forms co-precipitates with aggrecan, the main proteoglycan component of cartilage, and was previously found to be a good fixative in aiding the maintenance of chondrocyte morphology. We show that these RHT-aggrecan precipitates generate a positive chemographic signal on autoradiographic emulsions, in the absence of any radioactivity in the tissue section, via a process similar to the autometallographic process used previously for localization of trace metals ions in tissues. By exploiting the inherent depth-dependence of aggrecan concentration in adult articular cartilage, we demonstrated that the density of silver grains produced by RHT-derived chemography on autoradiographic emulsions correlated with locally measured aggrecan concentration as determined by the dimethylmethylene blue assay of microdissected tissue from these different depths of cartilage. To explore the benefits of this new method in monitoring tissue degradation, cartilage explants were degraded during culture using interleukin-1 (IL-1) or digested after culture using chondroitinase and keratinase. The RHT chemographic signal derived from these samples, compared to controls, showed sensitivity to loss of aggrecan and distinguished cell-mediated loss (IL-1) from degradation due to addition of exogenous enzymes. The RHT-derived chemographic grain density represents an interesting new quantitative tool for histological analysis of cartilage in physiology and in arthritis.     

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.     

A Selective Stain for Mitotic Figures, Particularly in the Developing Brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

A selective stain for mitotic figures, particularly in the developing brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

Simultaneous azo-coupling method for steroid acetate hydrolyzing enzyme

A simultaneous azo-coupling method for histochemical localization of steroid acetate hydrolyzing enzyme is described. It is based on the observation that d-equilenin, a natural oestrogenic steroid hormone, forms deeply coloured insoluble reaction products with diazonium salts under reaction conditions suitable for histochemical purposes. An acetate at position 3 of d-equilenin is rapidly hydrolysed by tissue esterase and the liberated d-equilenin couples with a diazonium salt to form a coloured precipitate. Steroid acetate hydrolyzing enzyme activity was observed in various tissues of the rat; a comparison with nonspecific esterase activity using alpha-naphthyl acetate as substrate suggested that steroid acetate hydrolyzing enzyme activity represents the activity of one or several isozymes of classical nonspecific esterase. This conclusion has also been drawn previously from biochemical studies using esters of other steroids.     

Histochemical demonstration of unsaturated hydrophilic lipids with palladium chloride.

Compounds in which olefinic linkages are accessible to aqueous reagents reduce the chloropalladite ion [PdCl4]2-, to metallic palladium. This reaction is used in a histochemical method whereby hydrophilic unsaturated lipids are stained dark brown or black. The specificity of the new method has been confirmed by means of solvent-extraction and chemical blocking procedures and by comparison with other histochemical techniques. Yellow staining of collagen, keratin and cytoplasm is probably due to attachment of the chloropalladite anion to proteins. The yellow background can be largely decolorized by treating the sections with aqueous pyridine, which forms colorless complexes with divalent palladium. A standard technique for staining with palladium is presented and the method is discussed in relation to other histochemical procedures that demonstrate unsaturated lipids.     

In situ studies of subcellular particles immobilized in sucrose-acrylamide density gradients.

In order to study subcellular particles. I have applied a method to immobilize the content of a sucrose density gradient with acrylamide after centrifugation. The different zones in the gels were localized and analyzed by specific stains and enzymatic, histochemical tests. This method allows the demonstration of several specific zones which could not be determined by traditional procedures. Moreover, the method requires only a few milligrams of material and can also be applied to other purposes such as autoradiography of gels containing minute amounts of radioactive products which could not be detected in a scintillation counter.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

Relationship among fibre type, myosin ATPase activity and contractile properties

Summary At least two types of skeletal muscle myosin have been described which differ in ATPase activity and stability in alkaline or acidic media. Differences in ATPase characteristics distinguish Type I and Type II fibres histochemically. In this study, ATPase activity of myosin from muscles of several species with known histochemical and contractile properties has been determined to test the hypothesis that (1) myosin ATPase activity, (2) histochemical determination of fibre types and (3) maximum shortening velocity, all provide equivalent estimates of contractile properties in muscles of mixed fibre types. Maximum shortening velocity appears to be proportional to ATPase activity as expected from previous reports by Barany. However, both myosin ATPase and the maximum shortening velocity exhibit curvilinear relationships to the fraction of cross-sectional area occupied by Type II fibres. Therefore, we reject the hypothesis and conclude that histochemically determined myofibrillar ATPase does not accurately reflect the intrinsic ATPase activity or shortening velocity in muscles of mixed fibre types. Our data are consistent with the presence of more than two myosin isozymes or with a mixture of isozymes within single muscle fibres.     

Histochemistry: historical development and current use in pathology

We describe the history of the histochemical stains that contributed most to the development of modern pathology during the last two centuries. Histochemical stains are presented in a list, which provides the essential information about year, country and main use of each to enable the reader to follow the chronological and geographical history of histochemistry. In addition to the historical evaluation of histochemistry development, we investigate how many classical histochemical stains survive in a modern laboratory of pathology and how often they are used for diagnostic practice compared to immunohistochemical (IHC) techniques. A ratio of about one histochemical reaction to 13 IHC reactions was tabulated. Finally, our data make it possible to define different cultural approaches to the terminology of histochemical and IHC stains: the former were based on eponyms, which link the stain with the name of its inventor, while the latter use a more impersonal biological terminology.     

A histochemical investigation of the mechanism of Aldehyde Fuchsin staining of pancreatic B-cell granules

Summary The reaction mechanism by which Aldehyde Fuchsin selectively stains pancreatic B-cell granules is unknown. The participation of either insulin or proinsulin in the reaction is debatable; the stain may be bound by other components of the B-cell granule or its membrane. Sections of pancreas were stained with a variety of basic stains and specific histochemical reagents with and without appropriate blocking agents. No evidence for strong tissue anions associated with the B-cell granule could be found. Aldehyde Fuchsin staining was not abolished by lowering the pH below the point at which all known tissue anions should be protonated. There was no evidence that the Aldehyde Fuchsin staining solution itself generates reactive groups in the tissue. The results of this investigation support a non-ionic, possibly covalent mechanism for Aldehyde Fuchsin staining of pancreatic B-cell granules.     

A Modified Freezing-Drying Apparatus for Tissues

The preparation of tissues by the freezing-drying technic is preferred for many histochemical studies because of the rapid fixation, avoidance of deleterious action of chemical fixation and extraction by aqueous and lipid solvents in fixation, dehydration and clearing; to facilitate this procedure a freezing-drying apparatus has been constructed which permits cutting of sections within five hours after the fresh tissue is obtained. Liquid nitrogen provides a most efficient moisture trap and in conjunction with a heating element provides any desired temperature down to — 80°C. during tissue drying. Paraffin infiltration is started without disassembling the equipment and completed in a few minutes. Most stains and histochemical reactions for enzyme and other substances can be applied directly to sections of frozen-dried tissue cut from the same blocks.     

High-performance thin-layer chromatography-bioautography for multiple antibiotic residues in cow's milk

An analytical method to identify and quantify multiple antibiotic residues (chloramphenicol, ampicillin, benzylpenicillin, dicloxacillin and erythromycin) in cow's milk by high-performance thin-layer chromatography (HPTLC) combined with bioautography was developed. The test microorganism used for bioautography was Bacillus subtilis ATCC 6633. Antibiotic residues were extracted with acetonitrile, fat eliminated with petroleum ether and residues isolated with dichloromethane The sensitivity of the method guarantees the detection of the above-mentioned antibiotics at levels below maximum residue limits (MRL) allowed for milk. Percentage recoveries ranged between 90 and 100%, with coefficients of variation between 7.2 and 21.3%. Some advantages of this methodology over thin-layer chromatography (TLC)/bioautography are also discussed.