bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochemical localization of glucose-6-phosphatase in rabbit mononuclear phagocytes during differentiation.
Authors: Nichols B A , Setzer P Y
Published: April 2011

Cytochemical and biochemical investigations have revealed glucose-6-phosphatase (G-6-Pase) activity in Kupffer cells of the liver. To determine whether other mononuclear phagocytes are also reactive for G-6-Pase, rabbit bone marrow, blood, and alveolar macrophages were tested for G-6-Pase by a modified Wachstein-Meisel method and prepared for electron microscopy. Some mononuclear phagocytes from all three tissues were intensely reactive; others were unreactive. In promonocytes, monocytes, and alveolar macrophages, reaction product for the enzyme was localized throughout all cisternae of the endoplasmic reticulum (ER) and the perinuclear cisternae, but it was absent from the Golgi complex, lysosomes, and occasional smooth tubular channels. These results indicate that mononuclear phagocytes at all stages of development contain cytochemically demonstrable G-6-Pase and that the distribution of the enzyme is not altered during their differentiation from immature cells in the bone marrow to mature macrophages in the lung.
A simple DNA-dependent fluorescence enhancement assay for cell number.
Authors: Adams C J , Storrie B
Published: April 2011

A DNA-dependent fluorescence enhancement assay using the dye Hoechst 33258 was developed to quantitate cell number in crude detergent lysates of cells. The assay, which uses a simple filter fluorometer, is sensitive enough to measure 1 x 10(4) cells without interference from mercaptans, culture media, and protein solutions. They assay can be done with freeze-thawed lysates. Fluorescence intensity values are linearly related to cell number in the range of 1 x 10(4) to 2.5 x 10(5) cells for L-929, BHK, and CHO-Kl cell lines.
A proteolytic procoagulant associated with malignant transformation
Authors: Gordon SG
Published: April 2011

There is increased deposition and lysis of fibrin in cancer, but little is known about the causes of this abnormal fibrin formation. This article reviews research on cancer procoagulant and presents some preliminary immunohistochemical studies. Cancer procoagulant was purified from a rabbit V2 carcinoma. It was a single polypeptide cysteine protease with a molecular weight of 68,000 daltons and an isoelectric point of 4.8-4.9. It initiates coagulation by directly activating factor X within the coagulation cascade. Its physical, chemical, and enzymatic properties distinguish it from serine proteases within the coagulation cascade and suggest that it is a protein from neoplastic cells. To determine whether cancer procoagulant was unique to the malignant state, procoagulant activity was assayed in extracts of normal and malignant tissue and serum-free medium from normal and transformed fibroblasts. Normal tissue and cells had procoagulant activity with characteristics similar to tissue factor, while malignant tissue and transformed cells had a procoagulant with the characteristics of cancer procoagulant, suggesting that cancer procoagulant is unique to the malignant state. An antibody to cancer procoagulant was used for horseradish peroxidase immunohistochemical staining fo cultured rabbit V2 carcinoma cells. These preliminary results support the concept that malignant cells produce the cancer procoagulant antigen.
Effects of glutaraldehyde and osmium tetroxide on newborn rat epidermal keratin.
Authors: Tzeng S , Fukuyama K , Epstein W L
Published: April 2011

The effects of glutaraldehyde and osmium tetroxide (OsO4) fixation on ultrastructural and biochemical changes of epidermal keratin were investigated. Morphometrically, the apparent size of keratohyalin granules was smallest when granular cells were fixed with OsO4, whereas those fixed with glutaraldehyde were larger, and those double-fixed with glutaraldehyde and OsO4 were largest. The ultrastructure of tonofilaments appeared different in each type of fixation. Biochemically, cross-linking glutaraldehyde with purified keratin from cornified cells formed a high molecular weight product with a blue shift of the absorption maximum and an increase in absorbance. Detection of the absorption spectra of OsO4- keratin reactions revealed that a new absorption maximum around 410 nm developed in glutaraldehyde-treated keratin, while one around 315 nm appeared in nontreated keratin. In addition, glutaraldehyde pretreatment increased by 40% the initial reaction rate and by 23% the total amount of OsO4 reduction. Sodium dodecyl sulfate gel electrophoresis demonstrated that degradation of keratin molecules occurred in both glutaraldehyde-treated and untreated keratin after 2 hr of exposure to OsO4. These findings indicate that keratin molecules are chemically modified by fixatives routinely used for electron microscopy. We consider that this chemical information plays an essential role in the appropriate interpretation of the ultrastructure of epidermal cells.
Effects of thyroidectomy, propylthiouracil, and thyroxine on pituitary content and immunocytochemical staining of thyrotropin (TSH) and thyrotropin releasing hormone (TRH).
Authors: Childs G V , Yang H Y , Tobin R B , Wilber J F , Kubek M
Published: April 2011

Previous studies have demonstrated immunocytochemical staining for beta chains of thyroid stimulating hormone (TSH-beta) in rough endoplasmic reticulum of pituitary cells hypertrophied after thyroidectomy ("thyroidectomy cells") (Moriarty CG(1976): J Histochem Cytochem (24:846; Moriarty GC, Tobin RB (1976): J Histochem Cytochem 24:1140). Here we report the localization of thyrotropin releasing hormone (TRH) in serial sections of the same pituitaries to determine if it could be found at similar sites. No staining for TRH was found in hypertrophied TSH cells formed 42 days after the surgery, or after 14, 34, and 70 days of propylthiouracil (PTU) treatment. The loss in immunostaining in the PTU-treated rats was correlated with radioimmunoassay (RIA) measurements that showed a 65% reduction in anterior pituitary TRH content after 34, 70, and 98 days of PTU treatment (from 22.9--7.8 pg/mg wet wt) and a 50% reduction in TSH content after 34 days of treatment. When thyroxine was administered to hypothyroid rats for 3 days before death, our previous studies had demonstrated intense staining for TSH in granules inside the rough endoplasmic reticulum. In this study, the radioimmunoassay showed that TSH content rose dramatically in the hypothyroid animals treated with PTU for 77 days and thyroxine for 2 days before death (from 8.5--64.1 mU/mg wet wt); however, the rise in TRH content was minimal (5.8--9.8 pg/mg wet wt). The immunocytochemical stain for TRH correlated well with the RIA showing a weak reaction mainly on small granules in the cytoplasm. No reaction for TRH was found in rough endoplasmic reticulum. These results suggest that TRH and TSH storage sites are dissimilar in the hypothyroid rat. The presence of stain for TRH in granules in the cytoplasm suggests that it might play a role in the storage or packaging of TSH. Its absence in profiles of rough endoplasmic reticulum staining intensely for TSH suggests that it is not synthesized at this site. No definite conclusions about its origin can be drawn at this time.
Spectral properties of fluorescence induced by glutaraldehyde fixation.
Authors: Collins J S , Goldsmith T H
Published: April 2011

Following fixation with glutaraldehyde, tissues or gelatin films fluoresce. This fluorescence can be enhanced more than thirtyfold by several minutes exposure to near ultraviolet light. Longer wavelengths produce a smaller effect. The enhanced fluorescence is maximally excited at 540 nm (half-band width about 45 nm), and fluorescence emission peaks at 560 nm. The rate of photoenhancement is independent of temperature in the 12--30 degrees C range. Photoenhancement is greater at alkaline than acid pH; the pH dependence involves a single acid binding group with pK = 7.3. These effects are not observed following treatment with paraformaldehyde or prior reduction with boranedimethylamine. Schiff base linkages between the bifunctional cross-linking reagent and free amino groups therefore seem to be involved. The effects of pH and wavelength on the photoenhancement of fluorescence are best accounted for by a kinetic scheme that includes both photogeneration of fluorophore from an ultraviolet-absorbing precursor, and its subsequent photodestruction by visible light.
Development of a procedure for autoradiographic localization of carbonic anhydrase at the electron microscope level.
Authors: Gay C V , Shanabrook V M , Sharkey D J
Published: April 2011

A method for preparing tissue suitable for electron microscope autoradiographic localization of carbonic anhydrase is described. Radioactivity is in the form of 3H-acetazolamide, a specific inhibitor of carbonic anhydrase. Tissues fixed in glutaraldehyde, dehydrated in ethylene glycol followed by cellosolve as a transition fluid, and embedded in epoxy resin, were found to retain most (74%) of the label. Electron micrographs of avian gastric mucosa prepared in this manner are shown. Other methods of preparation were explored and resulted in considerable losses of label or in inadequately preserved tissue. Light microscope autoradiographic localization of gastric mucosa, shell gland, chorioallantoic membrane, and skeletal muscle compare well with previous localizations.
A novel immunocytochemical model system for specificity and sensitivity screening of antisera against multiple antigens.
Authors: Larsson L I
Published: April 2011

A novel, convenient immunocytochemical model system that allows simultaneous testing of multiple peptides and peptide fragments is presented. Screening of antisera for specificity and sensitivity and ready detection of multiple antibody subpopulations is possible with this system. Using the peroxidase-antiperoxidase method, a threshold sensitivity of 100 pg of peptide was attained with the best antisera available. Routine screenings of antisera employ amounts of peptides corresponding to 1.8--180 pmol--concentrations about a thousandfold lower than those used for absorption controls. Because of the minute amounts of antigens needed, specificity screening against rare or costly antigens now becomes feasible.
Localization of DNA phosphorus in Tipula iridescent virus by oxygen plasma microincineration. I. Electron microscopy of ash structures.
Authors: Thomas R S
Published: April 2011

Tipula iridescent virus (TIV) is a relatively large particle containing about 15% DNA. As shown elsewhere by X-ray microanalysis (Thomas RS, Corlett M: J Histochem Cytochem 29:394, 1981), low-temperature oxygen plasma microincineration of the virus produces a stable phosphorus oxide ash representing this DNA nearly quantitatively. Transmission electron microscopy (TEM) of entire particles after plasma incineration shows the ash confined to the viral cores, confirming the previously known general location of the nucleic acid. Examination of ultrathin-sectioned virus crystals after plasma etching or ashing shows, on a still finer scale, that the DNA is probably confined to a shell structure within the core. A fine trace of ash from the capsid, seen in some preparations, may represent a phospholipid internal membrane known to be present. The possibilities of ash pattern artifacts are discussed. Heating experiments show that the ash patterns (and native virus particles) evaporate completely at high temperature, consistent with their presumed polyphosphoric acid composition. A heat-stable ash could be formed, however, when the viral DNA became accidentally stained with iron from the steel TEM grids used--a noteworthy artifact. The present work suggests some future possibilities of the plasma microincineration technique. In particular, the ability to see directly the fine distribution of mineral concentrations in ash patterns with the full resolution of TEM should be a powerful adjunct to increase effectively the sensitivity and resolution of X-ray microanalysis of mineral constituents in biological specimens.
Evaluation of periodate/lysine/paraformaldehyde fixation as a method for cross-linking plasma membrane glycoproteins.
Authors: Hixson D C , Yep J M , Glenney J R , Hayes T , Walborg E F
Published: April 2011

Fixation by periodate/lysine/paraformaldehyde, a method purported to cross-link specifically plasma membrane glycoproteins, was evaluated using Novikoff rat ascites hepatocellular carcinoma cells. Cells were treated with periodate/lysine, periodate/glycine, and periodate/lysine/paraformaldehyde and subsequently reduced with NaB3H4. The glycoproteins labeled with 3H were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by fluorography. The effects of reactant concentrations on 3H-labeling of cellular components, cell viability, and cross-linkage of 3H-labeled proteins were examined. The effect of increasing the localized density of plasma membrane glycoproteins on the extent of cross-linkage by periodate and lysine was investigated using cells in which patching of the plasma membrane glycoproteins had been induced by ferritin-conjugated concanavalin A/rabbit antiferritin antiserum. Also investigated was the periodate-independent to mixtures of periodate and lysine or glycine. Results of these studies did not support a mechanism of cross-linking involving reaction between the free base lysin and aldehyde groups on periodate oxidized carbohydrate residues but suggested a complex interaction between periodate oxidized plasma membrane glycoproteins and polymeric complexes of lysine and formaldehyde.U
Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.
Authors: Roth J , Bendayan M , Carlemalm E , Villiger W , Garavito M
Published: April 2011

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.
Immunofluorescent localization of cathepsins B and D in human fibroblasts.
Authors: Mort J S , Poole A R , Decker R S
Published: April 2011

The proteinase cathepsin B has been localized in human fibroblasts using a monospecific antiserum that reacts in a precipitating manner with the denatured enzyme only. The distribution of staining as revealed by both direct and indirect immunofluorescence indicates a typical lysosomal localization similar to that observed for cathepsin D. Using dual localization methods employing fluorescein- and tetramethylrhodamine-labeled affinity purified antibodies both proteinases were usually observed in the same lysosome, indicating a combined role in intracellular digestion.
Kinetic, computer, and electron microscopic studies dealing with an artificial enzyme membrane: theoretical and experimental evaluation of the cytochemical demonstration of acetylcholinesterase.
Authors: Malpiece Y , Sharan M , Barbotin J N , Personne P , Thomas D
Published: April 2011

The cytochemical demonstration of acetylcholinesterase is theoretically and experimentally evaluated with a model system. The model is an artificial proteic membrane in which acetylcholinesterase homogeneously is immobilized chemically by a bifunctional agent, glutaraldehyde. The copper-thiocholine histochemical method is studied kinetically and the system is simulated by computer calculations based on experimental kinetic parameters and numerical analysis methods. In addition, the corresponding electron micrographs are presented. These studies lead to the conclusions that the system is ruled by diffusional constraints and that enzyme distribution and repartition of the insoluble electron dense product are not circumscribed by any specific conditions.
Unlabeled antibody peroxidase-antiperoxidase method combined with direct immunofluorescence.
Authors: Valnes K , Brandtzaeg P
Published: April 2011

Paired staining with the unlabeled antibody peroxidase-antiperoxidase (PAP) method and direct immunofluorescence (DIF) differentiated distinctly between gastrin- and somatostatin-producing cells in the human gastric antrum. Similar paired staining of complexed lambda and alpha chains in immunoglobulin (Ig)A myeloma cells, of kappa and free J chains in IgG myeloma cells, and of secretory IgA and its epithelial transport protein, the free secretory component (SC), in colonic crypt cells, demonstrated that PAP staining inhibits subsequent DIF staining of an antigenic determinant present on the same molecule as the antigen revealed by the brown color of diaminobenzidine (DAB) or present or an unassociated molecule in the same cell. A quenching effect of the DAB reaction product was noted for both fluorescein (green) and rhodamine (red) emissions. In addition, a blocking effect of the DAB deposits has been demonstrated and is assumed to be the principal methodological basis for the paired PAP-DIF staining approach omitting intermediate antibody elution, as well as for the more time-consuming sequential PAP staining with DAB substrate for the first and 4-chloro-1-naphthol (CN) for the second antigen. The quenching and blocking effects limit in practice the paired PAP-DIF method to the localization of antigens present in separate cells.
Localization of low molecular weight protease inhibitor in serous secretory cells of the respiratory tract.
Authors: Kramps J A , Franken C , Meijer C J , Dijkman J H
Published: April 2011

We prepared in rabbits an antiserum against low molecular weight protease inhibitor (LMI) purified from the sputum of patients with purulent bronchitis. Using this antiserum in an immunoperoxidase staining method we found that this inhibitor was located exclusively in the serous cells of the submucosal glands of human upper and lower airways. The inhibitor was localized also in serous cells of the sublingual and submandibular glands. In contrast, LMI could not be demonstrated in the serous cells of the parotid gland. In the tissues investigated a strong association between the localization of the protease inhibitor and lysozyme was observed. Our observations indicate that the inhibitor may be present together with lysozyme as a secretory product in the serous cell granules. The possible consequences of the coexistence of these two proteins in the defense mechanism of the respiratory tract is discussed.
Microphotometric analysis of protein-bound thiols and disulfides with an azogenic maleimide.
Authors: Wiese D A , Sippel T O
Published: April 2011

The chromogenic thiol reagent N-(4-aminophenyl)-maleimide (APM) was used to provide a sensitive procedure for the determination of protein-bound thiol group, both before and after reduction of disulfide bonds by tri-n-butylphosphine. The bound reagent is diazotized and coupled at low pH to N-(1-naphthyl)ethylenediamine to form an intense magenta label (lambda max 547 nm) with a high molar absorbancy (epsilon 547 = 49,800 cm2/mmol). The validity of absolute quantitation afforded by the method was shown by comparing the results with standard chemical analyses of thiol contents in a model system. Furthermore, the applicability of the procedure to biological objects was shown on isolated epididymal bull sperm heads.
Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase.
Authors: Berod A , Hartman B K , Pujol J F
Published: April 2011

Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. Thus, the optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. Unless the results of localization using such a series is available, one must be satisfied with adequate but less than optimal results. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was shown to be highly pH dependent. By perfusing the tissue with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This two-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the second fixative solution. The results obtained by increasing the pH of the second solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde maybe obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the second step.
Postembedding staining of Brunner's gland with lectin-ferritin conjugates.
Authors: Suzuki S , Tsuyama S , Suganuma T , Yamamoto N , Murata F
Published: April 2011

Postembedding staining of intracellular carbohydrates of rat Brunner's gland cells embedded in Epon and acrylamide was carried out with Ricinus communis agglutinin-ferritin, concanavalin A-ferritin, and wheat germ agglutinin-ferritin conjugates. Th Golgi vacuoles and mucous granules were stained with these conjugates. In each staining, the tissues embedded in acrylamide were stained more strongly than those embedded in Epon. The staining intensity of wheat germ agglutinin-ferritin was the strongest among the three conjugates and the staining intensity of Ricinus communis agglutinin-ferritin was stronger than that of concanavalin A-ferritin in both embedding methods. Free ferritin showed almost no binding to these structures and staining with the conjugates was inhibited by the addition of appropriate competitive sugars to the staining solutions. Osmium-postfixed tissues were not stained well with the conjugates. Washing of the sections with bovine serum albumin solution after staining was an essential step in the present method to reduce the nonspecific adsorption of the conjugates. The present method was very simple and had good reproducibility.
Mithramycin- and 4'-6-diamidino-2-phenylindole (DAPI)-DNA staining for fluorescence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles.
Authors: Coleman A W , Maguire M J , Coleman J R
Published: April 2011

The properties of two DNA-specific fluorochromes, 4'-6-diamidino-2-phenylindole (DAPI) and mithramycin, have been analyzed as reagents to quantitate cellular DNA by fluorescence microspectrophotometry. Optimal staining conditions and concentrations, and the effects of other cellular materials to which the dyes bind, have been evaluated in measurements of the DNA of rat, chick, and yeast nuclei, Gonyostomum chloroplasts, and T4 particles. Use of either fluorochrome permits a high degree of resolution of different DNA quantities in nuclei and in cell organelles, and the DAPI-DNA complex is sufficiently fluorescent to permit quantitation of the DNA content in genomes as small as those of individual T4 bacteriophage particles. Fluorescence of mithramycin- or DAPI-stained DNA is proportional to DNA quantity when DNA of the same has composition is compared. Quantitation does not appear to be affected discernably by the state of the DNA, whether in different stages of the cell cycle, in condensed chromosomes, or in noncycling, differentiated nuclei. The use of chicken red blood cells is recommended as an internal monitor for variations in staining conditions.
Comparison of horseradish peroxidase visualization methods: quantitative results and further technical specifics.
Authors: Morrell J I , Greenberger L M , Pfaff D W
Published: April 2011

Four methods used for the neurohistochemical demonstration of horseradish peroxidase (HRP) were quantitatively compared by counting retrogradely labeled neurons found after each method was used. HRP used as a retrograde marker is an important neuroanatomical tracing method, and maximum sensitivity in its demonstration of retrogradely, labeled neurons is important if these neuroanatomical studies are to completely demonstrate afferent neurons. The four methods compared were a diaminobenzidine (DAB) procedure, a Hanker-Yates procedure using P-phenylenediamine and pyrocatechol, an o-dianisidine procedure, and a tetramethyl benzidine (TMB) procedure. The TMB procedure resulted in a more complete topography of neurons afferent to the HRP application site, and demonstrated many more neurons in all afferent cell groups that either of the three other procedures. Use of the TMB method was especially critical in the cases of small HRP applications, a size useful for neuroanatomical studies, where the other methods demonstrated very few or no retrogradely labeled neurons. Neurons were judged to be retrogradely HRP labeled if they had small granules of the reaction product (the color varying with the chromogen) describing the somal shape, usually extending into the processes, and a clear nucleus. In addition, after the o-dianisidine or the TMB reaction a small number of retrogradely labeled neurons had soma and processes especially well filled with reaction product, giving the appearance of neurons from Golgi preparations. For a sensitive TMB reaction giving good results, exact H2O2 concentration, freshly prepared solutions, minimal postreaction exposure to alcohol, counterstaining, and clean glassware were each found to be important.
Primary fixation in osmium-potassium ferrocyanide: the staining of glycogen, glycoproteins, elastin, an intranuclear reticular structure, and intercisternal trabeculae.
Authors: Goldfischer S , Kress Y , Coltoff-Schiller B , Berman J
Published: April 2011

Primary fixation in an osmium-potassium ferrocyanide (K4Fe(CN)6) mixture combines selective fixation, staining, and extraction of various cellular components; membranes, glycogen, glycoproteins, and elastin are preserved and stained. An intranuclear reticular structure that is composed of 3-6 nm fibers and permeates the entire nucleus, except for the nuclear pores, is demonstrated by electron microscopic examination of tissues prepared in an osmium-potassium ferrocyanide fixative. Condensations of the reticulum parallel the distribution of heterochromatin in interphase nuclei. This preparative procedure also reveals a network of trabeculae that are associated with the cisternae of rough endoplasmic reticulum and connect the parallel cisternae in hepatocytes, plasmacytes, neurons, and pancreatic ancinar cells. The intercisternal trabeculae are associated with both free and bound ribosomes.
A new species of lysosome in rabbit polymorphonuclear leukocytes.
Authors: Shannon W A , Zellmer D M
Published: April 2011

A species of lysosome containing an acid hydrolase activity, i.e., acid trimetaphosphatase (TMPase) was demonstrated in rabbit polymorphonuclear leukocytes (PMNs). The lysosome was less reactive near neutral pH and non-reactive at alkaline pH. The structure appeared typically ovoid, unlike the "tubular lysosomes" reported by Oliver (J Histochem Cytochem 28:78, 1980) in several exocrine acinar cells and also observed in monocytes in this study. Neither was there Golgi activity, although there are probably few, if any, Golgi elements present in the mature neutrophil. It is interesting that the TMPase-containing lysosome occurs in an inclusion the size of a specific granule at a greater frequency than the nonspecific acid phosphatase-containing lysosome.
Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells.
Authors: Soligo D , de Harven E
Published: April 2011

White blood cells have been studied in the back-scattered electron imaging (BEI) mode of scanning electron microscopy (SEM) with cytochemical methods for endogenous peroxidase, acid phosphatase, and a silver-staining method for nuclei. Peroxidase-positive granules were seen with good contrast and resolution in myeloid precursor cells and acid phosphatase activity was easily detected in macrophages and monoblasts. Silver staining permitted recognition of the shapes and location of the nuclei. In spite of the cytochemical procedures, cell surface structures were reasonably well-preserved in all methods, making direct correlation of BEI and secondary electron imaging (SEI) images an attractive feature in cell research with the scanning electron microscope.
Reversibility of acid and alkaline deoxyribonuclease deficiency in malignant tumor cells.
Authors: Taper H S
Published: April 2011

The nature of DNAse deficiency, which appears to be characteristic for malignant tumor cells, was investigated by the histochemical lead nitrate technique under various experimental conditions. Reappearance of distinct alkaline and acid DNAse activity was observed on the periphery of spontaneously occurring tumor necrosis, at early stages of the in vitro induced tumor necrosis, in necrotic tumor cells after in vivo irradiation and after in vitro treatment with different compounds. A membrane releaser did not reactivate DNAses in viable tumor cells, whereas the homogenate from tumor tissue inhibited DNAses in normal rat liver. These findings indicate that alkaline and acid DNAse deficiency in malignant tumor cells is a reversible phenomenon. This reversal of enzymatic activity has different histochemical and chronological patterns and specific reactivating factors for each DNAse. The masking effect of DNAse activity in malignant tumor cells is probably linked to natural enzyme inhibitors and its reversal to early stages of tumor necrosis.
HISTOCHEMICAL DEMONSTRATION OF NORADRENALINE AND ADRENALINE IN THE ADRENAL MEDULLA
Authors: HILLARP NILS-ÅKE , HÖKFELT BERNT
Published: April 2011

The optimal conditions for the histochemical demonstration of noradrenaline in the adrenal medulla by selective pigment formation at potassium iodate oxidation have been investigated. In addition the classical chromaffine reaction has been modified for the simultaneous demonstration of adrenaline and noradrenaline.
A METHOD FOR THE RAPID ESTIMATION OF WHOLE BLOOD IN HOMOGENIZED TISSUE PREPARATIONS
Authors: GORDON MALCOLM W. , NURNBERGER JOHN I.
Published: April 2011

A simple, selective, quantitative method for the estimation of whole blood in homogenized tissue preparations is described. This method is based upon the differential absorption of hemoglobin and carbonyl hemoglobin. Recovery experiments from homogenates of rat kidney, heart, liver, and brain are presented.
BASOPHILIC CHROMIDIA AND MITOCHONDRIA IN NORMAL AND IN TUMOR TISSUE
Authors: FIALA SILVIO , SPROUL EDITH E. , BLUTINGER MARTIN E. , FIALA ANNA E.
Published: April 2011

1. Homogenized tissue mixed with Pyronin Y can be separated by differential centrifugation into red-stained nucleic acid-rich elements—nuclei and chromidia —and unstained nucleic acid-poor mitochondria. Pyronin can thus be useful in preparations of pure mitochondrial suspensions.

2. Chromidia thus obtained correspond to basophilic elements of cytoplasm observed in histological preparations. By electron microscopy, RNA content and sedimentation in gravitational field, they appear as a heterogenous population of granules of various sizes. Their RNA content (basophilic) appears to increase in proportion to decrease in their size and sedimentability.

3. Mitochondria of mouse liver and of certain mouse tumors appear to have a constant proportion of RNA to nitrogen (basophilic quotient). In rat pancreas a higher value was found.

4. Liver, pancreas and tumor tissue show completely different patterns in distribution of basophilia. Tumor tissue is characterized by scarcity of large granules (mitochondria and heavy chromidia), reduction in the amount of small chromidia (microsomes) and predominance of basophilia in non-sedimented smallest nucleoprotein granules. Tumors are thus distinguished from such tissues as pancreas in which mitochondria are also scarce but heavy and light chromidia abundant.

5. A considerable succinoxidase activity was invariably found in large chromidia. This activity was inhibited by incubation with crystalline ribonuclease.
THE METACHROMATIC STAINING REACTION
Authors: KRAMER H. , WINDRUM G. M.
Published: April 2011

I. Chromatographic and spectrophotometric studiés have been carried out on thionine, toluidine blue and azure A. Thionine contains blue and red components, in the ratio of approximately two parts of blue to one part of red. The blue fraction has strong metachromatic properties; the red component stains all tissues, particularly those which are basophilic, in a bright red colour. Toluidine blue and azure A both contain metachromatic and non-metachromatic fractions but there is no question of the metachromatic colours which they produce being due to differential staining as, unlike thionine, they contain no red fractions.

II. The dye concentrations and staining times necessary for optimum results have been studied and discussed.

III. The treatment of sections after staining, particularly the vexed question of alcoholic dehydration as opposed to the examination of wet sections, has been the subject of particular attention. It follows from our findings that when the metachromatic staining reaction is used for histochemical purposes it is essential that steps be taken to differentiate histochemically significant irreversible metachromasia from the non-specific reversible metachromasia which can be exhibited under certain conditions by an extremely wide range of tissue elements. This can be done either by mounting the sections after staining in Apáthy's medium or syrup of laevulose, as recommended by Lison (1935, 1936), but far more effectively by alcoholic dehydration.

IV. The findings of test tube experiments are not directly applicable to histological material in that although the metachromasia obtained on mixing solutions of metachromatic dyes and chromatrope substances is readily reversed by alcohol, the metachromasia seen histologically in sections of cartilage, mast cells etc., is never changed back to the orthochromatic colour by alcohol or drying.

V. In the light of the experimental findings a technique for carrying out the metachromatic staining reaction for histochemical purposes is suggested.
HISTOCHEMICAL OBSERVATIONS ON THE HARDERIAN GLAND OF THE ALBINO MOUSE
Authors: COHN SIDNEY A.
Published: April 2011

The Harderian gland is a compound tubulo-alveolar organ, apocrine in nature. In addition to its lipoidal secretion it usually contains a characteristic reddish-brown, porphyrin pigment.

The glandular cells and alveolar lumina contain phospholipids, triglycerides, and possibly cholesterol or cholesterol esters. Alkaline phosphatase activity is present in the vicinity of the basement membrane and basal portion of the glandular cells. The cytoplasm and nuclei of the cells are weakly basophilic. No demonstrable glycogen, acid mucopolysaccharides or iron are present.

The pigment is moderately to intensely sudanophilic and acid-haematein positive, is bleached by pyridine and mineral acids, contains no iron, is not dissolved by lipid solvents and is negative to the biuret test, xanthoproteic reaction, Millon reaction and the ninhydrin-Schiff reaction. It darkens with osmic acid and iron hematoxylin, reduces potassium ferricyanide, synthesizes indophenol and fluoresces with a bright red fluorescence.

The presence of a saliva-resistant, PAS-reactive material or a peracetic acid-Schiff reactive material within the glandular lumina may have resulted from the oxidation of unsaturated fats.

It is concluded that part of the lipid which the gland elaborates may be actually transformed into pigment or may be attached to the pigment as a lipoprotein complex.

Evidence obtained is interpreted as supporting the concept that the gland, instead of being a passive storage organ for pigment formed elsewhere in the body, is actively engaged in the production of this pigment.
ON THE HISTOCHEMISTRY OF THE HAIR, AS STUDIED WITH THE PERACETIC ACID-SCHIFF STAIN
Authors: FINDLAY G. H.
Published: April 2011

The peracetic acid-Schiff reaction was studied in the hair with a view to establishing its histochemical significance. Evidence was collected which favoured the view that the peracetic acid-Schiff reaction was not a general test for ethylenic linkages and that the Schiff reagent was not acting in its capacity as a specific reagent for aldehydes. The nature of this remarkable reaction was obscure, and consequently its histochemical validity at the present time seemed doubtful.
DISTRIBUTION OF AMYLOPHOSPHORYLASE IN VARIOUS TISSUES OF HUMAN AND MAMMALIAN ORGANS
Authors: TAKEUCHI TADAO , HIGASHI KUNIHIRO , WATANUKI SHENJIRO
Published: April 2011

Phosphorylase forms from glucose-1-phosphate in tissue sections a polysaccharide which agrees best with amylose, amylopectin and a lightly branched glycogen, judging from the reactions with iodine, the periodic acid Schiff method and Best's carmine stain.

Phosphorylase is demonstrated most intensely in striated skeletal, glossal and cardiac muscle, in tracheal and skeletal cartilage, in the smooth muscle of large and medium arteries, uterus, tubes, vagina, bladder etc., in liver cells and renal collecting tubules, sweat glands and their ducts, hair sheath cells, stratified squamous epithelia of epidermis, tongue, esophagus, cervix uteri and vagina, in some adrenal cortical cells and in nervous tissue.

The iodine reaction technique with activator and primer, is the most suitable for the histochemical demonstration of phosphorylase in animal tissues.
PITFALLS IN HISTOCHEMISTRY: AN UNSUCCESSFUL ATTEMPT TO DEMONSTRATE MUCOID SULFATASE ACTIVITY IN MAMMALIAN TISSUE
Authors: DOHLMAN CLAES-HENRIK , FRIEDENWALD JONAS S.
Published: April 2011

Attempts to demonstrate a sulfatase in mammalian tissues attacking sulfated mucopolysaccharides were unsuccessful. Both chemical and histochemical methods were employed.

Some risks involved in the use of the lead salt technique in enzyme histochemistry are pointed out.
Simultaneous Feulgen densitometry and autoradiographic grain counting with the Quantimet 720D image-analysis system. II. Automated grain counting.
Authors: Sklarew R J
Published: April 2011

A Quantimet 720 Image-Analysis System has been configured and programmed to enumerate silver grains over labeled nuclei in Feulgen-stained autoradiographs. The accuracy and reproducibility of the estimates have been documented in 3H-thymidine (3HTdR)-labeled rat kidney cell cultures. The Quantimet and visual grain count estimates showed excellent correlation over a wide range of counts and was independent of grain density and clustering pattern. One advantage of this approach is that the grain counts can be related to specific cellular structures. The simultaneous measurement of the mean optical density of the grain-free nucleus provides a way of evaluating 3HTdR-absorption effects for valid grain count comparisons. Using a light pen, about 800 cells are measured per hour. The grain count estimates may be made simultaneously with estimates of Feulgen-stained DNA content in the labeled nuclei. This automated technology opens new avenues for critical applications to cell cycle analysis and related problems.
Occurrence of alpha-fetoprotein-containing hepatocytes in human embryos and fetuses: an immunohistochemical study using the light and ultrastructural immunoperoxidase methods.
Authors: Nemoto N , Kawaoi A , Shikata T
Published: April 2011

The occurrence of alpha-fetoprotein (AFP)-containing hepatocytes in 12 human embryos and fetuses ranging from 30-32 days of estimated ovulation age (Streeter's horizon XIV) to 16-17 weeks of estimated menstrual age was investigated using the direct (horseradish peroxidase (HRP)-labeled anti-human AFP horse IgG(Fab')2) and/or indirect immunoperoxidase methods. Under light microscopy, the AFP-containing hepatocytes were successfully demonstrated in paraffin-embedded liver tissues fixed with 4% paraformaldehyde(PFA)-picric acid solution containing 0.5% glutaraldehyde (GA). As a result of the studies of the human fetal livers at different developmental stages, only a small number of AFP-containing hepatocytes were initially identified immunohistochemically at the stage of Streeter's horizon XIX. Ultrastructural immunohistochemistry (the block-staining method using anti-human AFP horse IgG(Fab')2) revealed that the immunoreactive products against AFP were demonstrated on the ribosomal granules of the rough endoplasmic reticulum (rER), outer nuclear envelopes, free ribosomes, and Golgi's apparatus, and also in the cisternal lumina of the ER. No reactive products were noted in the nuclei or mitochondria. Our observations confirmed the presence of AFP-producing ability of the hepatocytes and the ultrastructural localization of the sites of protein synthesis in the early stage of development of human livers. Furthermore, we describe the extreme usefulness of the block-staining method for demonstrating the subcellular localization of AFP using HRP-labeled reduced anti-AFP antisera on liver tissues fixed with 4% PFA-picric acid solution containing 0.5% GA.
Immunoperoxidase localization of albumin and fibrinogen in rat liver fixed by perfusion or immersion: effect of saponin on the intracellular penetration of labeled antibodies.
Authors: Pignal F , Maurice M , Feldmann G
Published: April 2011

Immunoperoxidase localization of albumin and fibrinogen in rat liver was tested with perfusion or immersion fixation and saponin as a membrane permeabilizing agent. The distribution of albumin- or fibrinogen-containing hepatocytes was examined by light microscopy. Labeled antibody penetration was assessed by electron microscopy on transversely cut cryostat sections. Paraformaldehyde liver fixation by perfusion, followed by incubation of the sections with labeled antibodies together with saponin, demonstrated that albumin and fibrinogen were present in all hepatocytes; mainly in the Golgi apparatus and rarely in the endoplasmic reticulum, the ultrathin sections being labeled throughout their entire thickness. A constant labeling of the endoplasmic reticulum was obtained when saponin was added from the beginning of fixation. In the absence of saponin, albumin was seen in most of the hepatocytes but only at the periphery of the transverse sections, in a few Golgi apparatus, and in some parts of the endoplasmic reticulum; under this condition, fibrinogen was not visualized in the hepatocytes. Paraformaldehyde liver fixation by immersion showed the presence of albumin or fibrinogen in a few hepatocytes only, with irregular labeled antibody penetration. The use of saponin did not improve albumin and fibrinogen localization, except when the liver was poorly fixed. These results show that liver fixation by perfusion gives a homogeneous labeling of all the hepatocytes, whereas fixation by immersion leads to a heterogeneous labeling. Satisfactory results are obtained with saponin, which must be used to improve the penetration of labeled antibodies when the liver is fixed by perfusion. Saponin does not work when immersion is employed, at least under the conditions tested.
Non-Hodgkin's lymphoma: identification of the monoclonal B lymphocyte component in the presence of polyclonal immunoglobulin.
Authors: Wood G W , Travers H
Published: April 2011

A high proportion of non-Hodgkin's lymphomas are neoplastic proliferations of B lymphocytes, and, as such, express integral membrane and/or cytoplasmic immunoglobulin (Ig). Because these cellular proliferations are monoclonal, the Ig of all neoplastic lymphocytes will have identical light chain type and idiotype. These tumors sometimes contain significant amounts of polyclonal Ig. In this study we demonstrate that the polyclonal non-B-lymphocyte-associated Ig may be removed by washing tissue at low pH to reveal either neoplastic B lymphocytes or neoplastic "null" lymphocytes. These observations should facilitate the application of immunohistology to the routine diagnosis of lymphoma.
Quantitative histochemical measurement of pyruvate and lactate in mouse oviduct during the estrous cycle.
Authors: Nieder G L , Corder C N
Published: April 2011

Pyruvate and lactate are important energy sources for preimplantation embryos cultured in vitro. The purpose of this study was to determine in vivo levels of these substances in mouse oviductal tissues throughout the estrous cycle. Quantitative histochemical assays were developed to analyze these metabolites in submicrogram samples of freeze-dried ampullar and isthmic oviduct. The potential for other alpha-keto acids to interfere with the pyruvate assay was assessed and found to be minimal with this procedure. The importance of the collection method in maintaining in vivo metabolite levels was demonstrated by the marked changes observed with extended anoxia or pentobarbital anesthesia. Pyruvate levels at 3 hr postovulation (2.6 mmol X kg-1 dry weight) were higher than 12 hr before ovulation or 12 to 72 hr after ovulation (1.6 to 2.2 mol X kg-1) in both ampulla and isthmus. Lactate levels were significantly increased at 12 to 24 hr in the isthmus (28 mmol X kg-1) compared to other times during the cycle (9-19 mmol X kg-1). The observed levels of these metabolites may reflect changes in oviductal metabolism, induced by the hormone pattern of the estrous cycle, that promote the availability of needed energy substrates for the early embryo.
Immunocytochemical localization of actin in the pancreatic exocrine cell.
Authors: Bendayan M , Marceau N , Beaudoin A R , Trifaró J M
Published: April 2011

Immunoreactive actin molecules have been localized in pancreatic acinar cells by the protein A-gold technique. The labeling was found at the level of the filamentous cell web, and in close association with the Golgi cisternae, condensing vacuoles, and zymogen granules delimiting membranes as well as with the plasma membrane. A weak labeling was also present over the dense content of the zymogen granules. The association of actin with different membranes implicates that contractile proteins might constitute structural membrane proteins and, thus, might play an important role in protein secretion.