bluuebunny/crossref_metadata_2025 huggingface dataset:
Topographical distribution of phospholipids in boar sperm plasma and intracellular membranes as revealed by freeze-fracture cytochemistry.
Authors: Kan F W , Lin Y
Published: April 2011

We used fracture-label and label-fracture cytochemistry in conjunction with the phospholipase A2-colloidal gold (PLA2-CG) technique to study the distribution of phospholipids in ejaculated boar spermatozoa. These techniques provide visualization of the topographical distribution of phospholipids in freeze-fractured sperm membranes in a three-dimensional view. In various freeze-fractured boar sperm membranes and crossfractured cytoplasmic structures, quantitative analysis revealed that the nuclear envelope membranes and the nuclear content possessed the highest labeling density of PLA2-CG. Moderate labeling was detected over acrosomal membranes, especially the inner acrosomal membrane. Replicas of both protoplasmic and exoplasmic fracture faces of the plasma membrane of boar sperm head showed a relatively low density of PLA2-CG labeling. Moreover, a differential distribution of phospholipids was seen over the protoplasmic face of the plasma membrane domains of the sperm head, which showed the highest concentration of gold particles in the postacrosomal region, followed by the equatorial segment and the anterior acrosome region. The PLA2-CG labeling densities over the post-acrosomal region and the equatorial segment were significantly higher than that over the anterior acrosome region. In the flagellum, an intense labeling was also seen over crossfractured mitochondria, dense fibers, and fibrous sheath. The protoplasmic fracture face of the plasma membrane over the middle piece, the annulus, and the principal piece was moderately labeled by PLA2-CG. No significant difference in mean labeling density of PLA2-CG was detected among the three membrane domains. In label-fracture preparations, exoplasmic halves of the plasma membrane of the head and the middle piece of the tail were uniformly labeled with PLA2-CG. However, the annulus and principal piece were devoid of PLA2-CG binding sites. These results indicate that differential distribution of phospholipids associated with the boar sperm membranes may reflect phospholipid composition of membrane domains characteristic of special physiological functions.
Distinct distribution of protein disulfide isomerase family proteins in rat tissues.
Authors: Iida K I , Miyaishi O , Iwata Y , Kozaki K I , Matsuyama M , Saga S
Published: April 2011

We investigated the distribution of protein disulfide isomerase (PDI) family proteins PDI, ERp72, and ERp61 in rat tissues and compared their localization by immunohistochemical double staining. The extent of their expression was diverse in different cell types, although they were ubiquitously distributed in a wide variety of cell types. Prominent staining for all three proteins was detected in thyroid follicular epithelia, tracheal mucous gland cells and chondrocytes, and chief cells and mucous neck cells of the stomach. Hepatocytes, neuronal cells in brain and spinal cord, and pancreatic acinar cells were also consistently and uniformly stained, but with low intensity. On the other hand, distinct differences were observed for the expression of the three proteins in plasma cells, pancreatic islet cells, goblet cells, and Paneth's cells of intestines, seminiferous epithelia, and salivary gland ductal epithelia. The similarities and differences in the distribution of the three proteins provide helpful clues to their biological functions.
Retrieval of leukocyte antigens in paraffin-embedded rat tissues.
Authors: Shetye J D , Scheynius A , Mellstedt H T , Biberfeld P
Published: April 2011

We compared the immunohistochemical reactivity of various rat leukocyte antigens in frozen and paraffin-embedded thymus, spleen, abdominal lymph node, liver, and brain tissues of healthy Sprague-Dawley rats, fixed in various fixatives. Immune reactivity after fixation in Methacarn was superior to that of 4% neutral buffered formalin, a mercury-based fixative (B-5), or Carnoy. Microwave (MW) antigen retrieval (AR) enhanced antigen reactivity. Ten of the 11 leukocyte antigens studied could be retrieved in Methacarn-fixed, paraffin-embedded sections with a reactivity comparable to that obtained on frozen sections.
A strategy for immunohistochemical signal enhancement by end-product amplification.
Authors: Chen B X , Szabolcs M J , Matsushima A Y , Erlanger B F
Published: April 2011

We report a novel strategy, called end-product (EP) amplification, capable of enhancing the sensitivity of immunohistochemical procedures by about an order of magnitude or more. The strategy employs an antibody (anti-EP) to the product generated by the action of horseradish peroxidase on 3,3'-diaminobenzidine (DAB), and can be extended to the products of other enzymes as well, e.g., alkaline phosphatase. Amplification is the consequence of the ability of anti-EP to detect the multiplicity of product moelcules resulting from the turnover of substrate by a single enzyme molecule. The subsequent detection of anti-EP was by biotinylated goat anti-rabbit antibody, followed by avidin-peroxidase and DAB or by avidin-alkaline phosphatase and Vector Red. Further amplification can be accomplished by repeated cycles of the protocol. Anti-EP was produced by immunization with a bovine serum albumin (BSA) conjugate of a soluble polymer of DAB, prepared by a carefully controlled reaction of DAB with horseradish peroxidase and hydrogen peroxide. Coupling to BSA (and to RSA) was accomplished with glutaraldehyde. The titer of anti-EP was established by ELISA. Formalin-fixed, paraffin-embedded sections of five cases of Hodgkin's disease and five tonsils with follicular hyperplasia were immunolabeled for the following lymphoid markers: CD3, CD20, CD30, CD45RA, and CD68. EP amplification with anti-EP was also applied to cases of CMV pneumonia and cerebral toxoplasmosis to determine whether this procedure could improve detection of the infectious agents. Immunolabeling of the primary antibody was performed by the avidin-biotin-peroxidase technique with DAB as the reaction substrate. The specificity of EP amplification was tested by demonstrating binding of anti-EP with Vector Red with the generation of a fluorescence end-point. There was complete congruence in the distribution of the DAB signal and the red immunofluorescence representing EP amplification. The intensity of the DAB signal was increased as much as 16-fold by EP amplification, making possible a reduction in the amount of the primary antibody by as much as 85-90%. Sensitivity also increased with respect to weakly expressed antigens and low concentrations of infectious agents.
The impact of fixatives on the binding of lectins to N-acetyl-glucosamine residues of human syncytiotrophoblast: a quantitative histochemical study.
Authors: Høyer P E , Kirkeby S
Published: April 2011

We describe a quantitative histochemical method for demonstration of five N-acetyl-glucosamine binding lectins in the syncytiotrophoblast of human term placenta. The method employs biotinylated lectins and alkaline phosphatase-conjugated avidin. The alkaline phosphatase activity is detected by using 5-bromo-4-chloro-indoxyl phosphate as the substrate and nitroblue tetrazolium as the capture agent. The effect of 13 fixative solutions on specific lectin binding and nonspecific background staining was quantified by microspectrophotometry. Acid fixatives or fixatives containing mercuric chloride, e.g., Carnoy's and Zenker's fixatives, gave intense specific lectin binding and low background staining. Glutaraldehyde, carbodiimide, and ethanol resulted in low specific lectin binding and a very high background staining that was mainly due to endogenous placental alkaline phosphatase. Lectin binding to N-acetyl-galactosamine, mannose, galactose, and fucose was also significantly higher in sections from tissues fixed in an acid fixative compared with a neutral buffered fixative. Unfixed cryosections revealed a considerably lower degree of specific lectin binding compared with sections from fixed tissues. The activity of endogenous placental alkaline phosphatase was inhibited dose-dependently by mercuric chloride and decreased with L-phenylalanine concentration over the range of 7.8 x 10(-4) M to 5 x 10(-2) M, after which there was no further inhibition. Calf intestinal-type alkaline phosphatase conjugated to avidin was not inhibited by 5 x 10(-2) M L-phenylalanine. Endogenous placental biotin did not contribute significantly to background staining. Despite the high level of placental alkaline phsophatase, the intestinal-type alkaline phosphatase can be used as a marker enzyme in the sensitive ABC technique, provided that the nonspecific background is measured and substracted. Moreover, it is advisable to use an acid- and/or mercuric chloride-containing fixative and to add L-phenylalanine during incubation steps.
Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.
Authors: Momayezi M , Wloga D , Kissmehl R , Plattner H , Jung G , Klumpp S , Schultz J E
Published: April 2011

We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.
Immunohistochemical localization of mouse Clara cell 10-KD protein using antibodies raised against the recombinant protein.
Authors: Ray M K , Wang G , Barrish J , Finegold M J , DeMayo F J
Published: April 2011

To investigate the developmental regulation of the mouse Clara cell 10-KD protein (mCC10), we raised an antibody against the recombinant mCC10 protein. The coding region for the mature mCC10 protein was placed in frame with the glutathione-S-transferase gene in the pGEX2-T bacterial expression vector. The GST-mCC10 fusion protein was expressed in E. coli DH5 alpha cells. The fusion protein was purified and eluted using glutathione-Sepharose beads. The GST-mCC10 fusion protein was injected into rabbits to raise antibodies. The rabbit anti-mCC10 antibody was tested by immunoblot analysis using both purified protein as well as extracts of lung, liver, and uterus. The antibodies produced were used in immunohistochemistry and immunoelectron microscopy to detect the cellular localization of this protein in the above organs. This anti-mCC10 antibody will be useful for future investigation of the developmental biology of the lung.
Synthesis of endothelin-1 in rat uterus during pregnancy.
Authors: Kajihara T , Tomioka Y , Hata T , Ghazizadeh M , Asano G
Published: April 2011

Endothelin is a potent vasoconstrictive peptide first isolated from the supernatant of cultured porcine aortic endothelial cells. The purpose of this study was to determine the source of endothelin-1 (ET-1) production in rat uterus during pregnancy and to clarify its role in normal pregnancy. ET-1, as recognized by immunohistochemistry, was weakly expressed only in endometrial glandular cells in nonpregnant rats but was intensely expressed in both glandular and myometrial cells in the early postpartum period. In situ hybridization confirmed the localization of prepro-ET-1 mRNA in the cytoplasm of endometrial glandular and myometrial cells but not in stromal cells or in smooth muscle cells of blood vessels. Northern blot analysis detected prepro-ET-1 mRNA in myometrial tissue from pregnant but not nonpregnant rats. In particular, the expression of prepro-ET-1 mRNA was strongest in the early postpartum period compared with the various stages of pregnancy. These results indicate that, in addition to endothelial cells and endometrial glandular cells, myometrial cells also produce ET-1 and its production significantly increases in the early postpartum period. Therefore, ET-1 may play a pivotal role in controlling bleeding from the placental bed through myometrial contraction.
In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.
Authors: Negoescu A , Lorimier P , Labat-Moleur F , Drouet C , Robert C , Guillermet C , Brambilla C , Brambilla E
Published: April 2011

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.
Immunocytochemical detection of regional protein changes in rat brain sections using computer-assisted image analysis.
Authors: Huang X , Chen S , Tietz E I
Published: April 2011

We used several approaches to assess the reliability and sensitivity of computer-assisted densitometry to detect regional changes in tissue antigen content as a function of immunohistochemical staining density. We designed a model system to mimic variations in antigen concentration in postfixed, slide-mounted rat brain sections by varying the ratios of conjugated (biotinylated) to unconjugated secondary antibody. Antigen concentration was also varied in tissue discs made from mixing rat brain homogenate with increasing amounts of tissue embedding compound. The monoclonal antibody bd-17 to the beta2/3 subunit of the GABAA receptor was used as the primary antibody. Immunostaining density was visualized with diaminobenzidine (DAB). There was a significant, positive linear relationship (r = 0.97-0.99) between immunostaining intensity and antigen concentration. With this approach, changes in antigen content of less than 10%, as reflected in immunostaining intensity, were detectable in brain sections. The low degree of variability in measures of regional variation in immunostaining in sections from naive rats (n = 7) suggested that the method was suitable for quantitative analysis and indicated the reliability of the method. This systematic study of the utility of computer-assisted image analysis for semiquantitative immunohistochemical analysis found the method to be both reliable and sensitive.
Expression of dopamine transporter at the tips of growing neurites of PC12 cells.
Authors: Kadota T , Yamaai T , Saito Y , Akita Y , Kawashima S , Moroi K , Inagaki N , Kadota K
Published: April 2011

The four kinds of oligopeptides specific in amino acid sequence to a rat dopamine transporter (DAT), peptide-1-peptide-4, were chemically synthethized. An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method. Two monoclonal antibodies, MAbs H-1a and H-1b, were produced against one of the oligopeptides, peptide-1. Western blot analysis confirmed that the two antibodies recognized an approximately 85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also confirmed by an antibody absorption test using two synthetic oligopeptides, one of which is specific only to DAT. These results have confirmed the specificity of the present antibody to DAT. The expression and subcellular localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF). The antibody labeled the surface of PC12 cells. When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endoplasmic reticulum and then on the surface of growth cones from the beginning of neurite outgrowth. DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [3H]-dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT, blocked the [3H]-dopamine at a concentration of 10(-7) M. The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased.
Differential distribution of galactosylceramide, H antigen, and carcinoembryonic antigen in rhesus macaque digestive mucosa.
Authors: Butor C , Couëdel-Courteille A , Guillet J G , Venet A
Published: April 2011

The most common route of transmission of HIV is via the mucosa. We compared human and macaque intestinal epithelia to determine whether the SIV macaque system can be used as a model to study HIV transmission by the rectal route. The overall morphology of the macaque gut mucosa is very similar to that of humans. Differentiation markers follow the same pattern as in the human system. The carcinoembryonic antigen (CEA) is apical in epithelial cells of the rectum and is absent from the small intestine. Blood group H antigen is expressed by enterocytes but not by colonocytes or rectocytes. Galactosylceramide, a potential alternative receptor for HIV in epithelial cells, is expressed in all intestinal segments as in humans. In absorptive cells it is apical and intracellular in the rectum, colon, and cecum, whereas it is only intracellular in small intestine. In goblet cells the galactosylceramide is present in intracellular vacuoles in all segments. It is also present on the membrane of mucous granules in colon and small intestine but not in rectum. We therefore believe that the macaque digestive tract may constitute a good model for the human digestive tract in the transmission of lentiviruses.
Immunochemical detection of arylamine N-acetyltransferase in normal and neoplastic bladder.
Authors: Stanley L A , Coroneos E , Cuff R , Hickman D , Ward A , Sim E
Published: April 2011

The N-acetyltransferase (NAT) phenotype is an important determinant of individual susceptibility to occupational bladder cancer. N-Acetyltransferases arc known to metabolize aromatic amine bladder carcinogens, but the functional significance of NAT expression in the target organ is unclear. To resolve this issue, polygonal antisera against purified recombinant enzymes and C-terminal peptides of human NAT Type 1 (NAT1) and Type 2 (NAT2) were generated. Western blot analysis of exfoliated cells from human urine, pig bladder homogenate, and human bladder tumor-derived cell lines showed that NAT1 was expressed in all three systems, whereas NAT2 did not appear to be expressed in the bladder. Immunohistochemical analysis of human bladder tumor sections indicated that well-differentiated tumor cells expressed NAT1, with the highest level of expression being found in the umbrella cells that line the bladder lumen. Poorly differentiated tumor regions appeared to express NAT1 at lower levels than did well-differentiated areas. These findings support the hypothesis that aromatic amines are metabolized in the bladder epithelium by NAT1.
In situ detection of individual transplanted bone marrow cells using FISH on sections of paraffin-embedded whole murine femurs.
Authors: Nilsson S K , Hulspas R , Weier H U , Quesenberry P J
Published: April 2011

Studies of transplantation biology rely on the detection of donor hemopoietic cells in transplant recipients. Traditionally this has been achieved through ex vivo techniques, including flow cytometric analysis of cell surface markers to detect cells expressing specific epitopes, histochemical detection of cytoplasmic proteins, and the detection of Y chromosome-specific sequences by DNA hybridization. Studies using congenic models, such as the Ly5.1/5.2 mouse, or the utilization of fluorescent dyes, such as PKH-26, have allowed more in-depth analysis of transplantation, beginning to address key issues such as cell homing through cell tracking and elucidation of the "stem cell niche." However, these methods are limited by labeling sensitivity, specificity, crossreactivity and, in the case of PKH-26 labeling, the number of cell divisions the transplanted cells can make before the signal disappears. We have developed a fluorescent in situ hybridization (FISH) technique that utilizes a murine Y chromosome-specific "painting" probe to identify in situ individual transplanted male cells in paraffin-embedded sections of female whole bone marrow while maintaining good morphological integrity. This method is highly sensitive and specific, labeling more than 99% of male cells and no female cells, allowing each transplant to be assessed at the individual cell level. The technique provides unique opportunities to follow the path taken by transplanted cells, both during homing into the marrow and through their maturation and differentiation into mature, functional hemopoietic cells.
AN ANALYSIS OF FROZEN SECTION TECHNIQUES: I. SECTIONING OF FRESH-FROZEN TISSUE
Authors: THORNBURG WAYNE , MENGERS PAUL E.
Published: April 2011

Optimal Conditions for cutting thin sections from fresh-frozen tissue have been described in terms of a set of optimal temperatures for the tissue block, microtome chamber, and knife. A model for the cutting process is proposed which postulates a zone of "micro-melting" at the edge of the knife. The behavior of the freezing microtome is interpreted in terms of this model for a variety of conditions.
THE ENTRY OF CA45 INTO THE SKIN AND OTHER SOFT TISSUES OF THE RAT: AN AUTORADIOGRAPHIC AND SPODOGRAPHIC STUDY
Authors: BÉLANGER LEONARD F.
Published: April 2011

The uptake of radiocalcium by specific regions of the skin, eye, cartilage and mucous glands has been demonstrated with the help of integrated autoradiographs.

Most of the calcium that enters the hair follicle is found in the keratogenous zone. In the skin and eye, it appears that Ca45 has entered by bio-synthesis while in the cartilage and mucous glands, that uptake of labelled Ca might represent cation adsorption by the sulfopolysaccharides.

The autoradiographic record of Ca45 follows the distribution of the blue/white dust in the spodograms of skin, cartilage and mucous glands.
OBSERVATIONS ON THE REDUCTION AND QUANTITATION OF NEOTETRAZOLIUM
Authors: BURTNER HELEN J. , BAHN R. C. , LONGLEY J. B.
Published: April 2011

1. By means of chromatography two different colored products and readily separated from reduced samples of neotetrazolium chloride.

2. Determinations have been made of the absorption spectra and specific absorption coefficients of these products.

3. It has been shown that the two products do not represent succeeding stages in the reduction of neotetrazolium, but must arise front different tetrazoles.

4. The significance of these findings with respect to the use of neotetrazolium in the quantitation of reducing activities and with respect to the histochemical usefulness of ditetrazoliums in general is discussed.
THE XANTHYDROL REACTION FOR PYRROLES AND INDOLES IN HISTOCHEMISTRY: ZYMOGEN GRANULES, LENS, ENTEROCHROMAFFIN AND MELANINS
Authors: LILLIE R. D.
Published: April 2011

Zymogen granules of the pancreas, peptic and salivary glands, eosinophil leucocyte granules, lens fibers, and sometimes Paneth cell granules give a violet color with xanthydrol. Muscle and various cytoplasms react weakly, collagen, clastin, keratin, chromaffin, enterochromaffin, melanin, bone, cartilage and mucus do not react.

The positive reaction seems largely assignable to the presence of tryptophan.

In vitro reactions occur both with various indoles and pyrroles and also with certain polyphenols.

The tissue reaction is attenuated and finally abolished by chromation and is inhibited by adequate iodination and by adequate peracetic acid oxidation.

Formaldehyde condensation with tryptamine only slightly weakens its reactivity to xanthydrol, and the positive reactions of carbazole and tetrahydronorharman also argue that formaldehyde should not prevent the reaction of 5-hydroxytryptamine to xanthydrol. Hence the identification of the enterochromaffin substance as such with 5-hydroxytryptamine seems increasedly improbable.
THE HISTOCHEMICAL DEMONSTRATION OF LEUCINE AMINOPEPTIDASE
Authors: NACHLAS MARVIN M. , CRAWFORD DAVID T. , SELIGMAN ARNOLD M.
Published: April 2011

1. A method is described for the histochemical localization of leucine aminopeptidase in fresh frozen and paraffin embedded tissues.

2. Evidence is offered which supports the idea that the substrate l-leucyl-β-naphthylamide is hydrolyzed by leucine aminopeptidase.

3. A comparison is made between the various diazonium salts used to demonstrate the enzyme in tissue sections. The shortcomings of each coupling agent are given. While the one recommended in this report has many favorable properties, its slow rate of coupling constitutes a serious drawback in localization at the cellular level. Experiments are in progress to overcome this deficiency in the method.

4. The value and future potential of chelations with metal ions in histochemical methodology is discussed.

5. Leucine aminopeptidase was found to be present histochemically in many organs of man, guinea pig, dog, and rat.
METAL REDUCTION REACTIONS OF THE MELANINS: HISTOCHEMICAL STUDIES
Authors: LILLIE R. D.
Published: April 2011

In general the melanins are argentaffin and, except in hair cortex, reduce ferric ferricyanide. Cutaneous and ocular melanins reduce acid silver nitrate, more promptly in guinea pigs than in man and rhesus monkeys. This reduction is slower than that of ammoniacal silver nitrate. It may be accelerated so as to occur in one or two minutes by prior reduction of the melanins by sodium hydrosulfite. Neuromelanin reduces acid silver nitrate very slowly (2-3 days) and prior hydrosulfite reduction does not induce rapid reductivity for acid silver. Acetylation has no influence on the reductivity of melanin for acid or ammoniacal silver or ferric ferricyanide. But, if applied to cutaneous and ocular melanins after reduction with hydrosulfite, it then greatly impairs the reduction of acid silver nitrate, but not of ammoniacal silver. Acetylation similarly influences the reductivity of catechol toward the acid and ammoniacal silver solutions.

Oxidation has two general effects on the melanins, an irreversible bleaching by the more drastic oxidants, and a reversible abolition or impairment of their reducing capacity toward acid and ammoniacal silver solutions and ferric ferri cyanide. The oxidants vary in their effect on the silver and ferric ferricyanide reactions, so that some are more effective against the argentaffin reactions, others against the ferric ferricyanide reaction. Likewise the cutaneous, ocular and nerve cell melanins show differences in loss of reductivity with the various oxidants.

Tumor melanins seem somewhat less reactive to silver than adjacent normal tissue melanins. Ocular tumor melanin resembles chorioidal melanin more than the epithelial melanins of the retina, ciliary and iris.

The argentaffin reaction of cutaneous and ocular melanin is probably assignable to the presence of quinhydrone groupings which may be reversibly oxidized to quinones and reduced to diphenols. This type of grouping does not appear to be present in neuromelanin. Neuromelanin, from this and from the other recorded differences, appears to be a distinct substance, perhaps not even closely related chemically to the other melanins.
ADAPTATION OF THE MOREL SISLEY PROTEIN DIAZOTIZATION PROCEDURE TO THE HISTOCHEMICAL DEMONSTRATION OF PROTEIN BOUND TYROSINE
Authors: LILLIE R. D.
Published: April 2011

Protein diazotization followed by alkaline coupling with a naphthol has been successfully applied for the histochemical localization of the protein bound tyrosine in tissues. Prolonged nitrous acid treatment in the cold is required (18-24 hours at 3°C). The best coupling reagent so far tried is 1-amimio-8-naphthol-4-sulfonic acid (S acid) and coupling succeeds best in a 1 % potassium hydroxide in 70 % alcohol, allowing 1 to 4 hours at 3°C. Topochemical results parallel those of the Millon reactions, with greater color intensities and superior histologic definition.
ON THE INCORPORATION OF METHIONINE 35S INTO PROTEINS DETECTABLE BY AUTORADIOGRAPHY
Authors: SIRLIN J. L.
Published: April 2011

As measured by autoradiographs, thioglycollate does not reduce the activity from sulphur in the proteins of the early chick embryo, if the sulphur has been presented as methionine. This indicates that the sulphur is peptide-bonded.
THE USEFULNESS OF THE BROMINATION VARIANT IN THE FEULGEN MICROSPECTROPHOTOMETRY
Authors: BARKA T. , DALLNER G.
Published: April 2011

The bromination Feulgen variant (T. Barka: J. Histochem. Cytochem. 4: 208, 1956) can be used even in the Feulgen microspectrophotometry. The decrease in UV-absorption and gallocyanin-chromalum staining caused by brominatoin suggest that the bromination depolymerizes but one part of the ribonucleic acid, beside that it releases aldehyde groups in the deoxyribonucleic acid.
HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN. XV. SITES OF PHOSPHORYLASE AND AMYLO-1,6-GLUCOSIDASE ACTIVITY
Authors: ELLIS RICHARD A. , MONTAGNA WILLIAM
Published: April 2011

The localization of phosphorylase and amylo-1,6-glucosidase activity has been studied in surgical specimens of human skin from the palm, sole, axilla, external auditory meatus, and other representative regions of the body. With few exceptions these enzymes are found in cells which are known to contain glycogen normally. The epidermis shows some variability, but amylo-1,6-glucosidase is generally present in the stratum spinosum, while phosphorylase is found in both the stratum basale and the stratum spinosum. The relative amounts of the enzymes vary with the thickness of the epidermis and with the age of the donor. Growing hair follicles have abundant phosporylase and amylo-1,6-glucosidase in their outer root sheaths, while resting ones contain only phosphorylase. A short portion of the epidermal duct of the eccrine sweat glands has no enzymatic activity, but the remainder of the duct and the secretory portion of the gland is richer in phosphorylase than any other structure of the skin. The apocrine sweat glands have neither enzyme in their secretory coils, but the duct of these glands is rich in phosphorylase. Time sebaceous glands contain both enzymes, but phosphorylase is more concentrated in the peripheral cells of the gland. Neither the centers of the glands nor the sebum contain either enzyme.
HISTOCHEMICAL DEMONSTRATION OF BRANCHING ENZYME (AMYLO-1,4 →1,6-TRANSGLUCOSIDASE) IN ANIMAL TISSUES
Authors: TAKEUCHI TADAO
Published: April 2011

The procedure for the histochemical demonstration of branching enzyme (amylo-1,4 → 1,6-transglucosidase) activity is given. Procedures to determine the reliability of the results of localization of enzymatic activity and the histochemical relation between branching enzyme and amylophosphorylase are discussed. The results in normal tissues of the rabbit are presented. The distribution of amylo-1,4 → 1,6-transglucosidase in animal tissue cells is quite similar to that of amylophosphorylase. The quantitative balance of each enzyme in tissue cells is changed respectively according to various conditions. Greatest concentration of enzymatic activity is seen in liver, skeletal muscle, heart muscle, smooth muscle of various organs, nervous tissues and epithelium of certain organs.
CERTAIN TECHNICAL ASPECTS OF FLUORESCENCE MICROSCOPY AND THE COONS FLUORESCENT ANTIBODY TECHNIQUE
Authors: TOBIE JOHN E.
Published: April 2011

A fluorescence microscope possessing an efficient ultraviolet light source, the 1000 watt A-H6, is described with special reference to its application in studies concerned with the fluorescent antibody technique and the tissue localization of the tetracyclines. The spectral characteristics of the light source are discussed and methods outlined whereby combinations of certain ultraviolet transmitting and absorbing filters can be used for maximum stimulation and visualization of fluorescent substances in tissue specimens. In fluorescent antibody studies when fluorescein isocyanate is used as the fluor, superior results have been obtained by excitation of tissue sections with blue-violet radiation at a wavelength band of 390-440 mµ rather than with ultraviolet radiation at 360-370 mµ. The two outstanding advantages of the longer wavelength stimulation are: 1) The increased fluorescence and consequent visual enhancement; and 2) the greater cellular detail for visual orientation and photographic clarity thus obtained. In contrast to this, the tetracyclines were more effectively stimulated in sections of bone with ultraviolet radiation at wavelengths in the 360-370 mµ band.

A method of quick-freezing fresh tissues in petroleum ether at approximately –65° C has been found more satisfactory for preserving the cellular architecture of tissues used in the fluorescent antibody technique. The operation of a rotary microtome in a cryostat at sub-freezing temperatures has been improved by special lubrication with the result that sections can be cut at a microtome setting of 3 µ. With these thin sections, it is possible to observe more minute cytological details and to localize fluorescent substances more accurately.
DYEBINDING AND MUCOPOLYSACCHARIDE CONTENT IN CONNECTIVE TISSUE
Authors: BALAZS ENDRE A. , SZIRMAI JOHN A.
Published: April 2011

The hyaluronic acid content of the mucoid layer of the rooster comb and the chondroitin sulfuric acid content of bovine nasal cartilage were determined in the fresh tissue, in the water extract of the tissue, and in the water-extracted residue. The binding of cationic dye by the tissue, the tissue extract and the residue was compared with the mucopolysaccharide content. The molar ratio of the bound dye to the anionic sites of the mucopolysaccharides varied between 1.1 and 1.8. In the comb extract this ratio was one. This indicates that, with the exception of the comb extract, anionic sites other than those of the mucopolysaccharides are also responsible for the dyebinding.
THE HISTOCHEMICAL DEMONSTRATION OF CYTOCHROME OXIDASE WITH A NEW REAGENT FOR THE NADI REACTION
Authors: NACHLAS MARVIN M. , CRAWFORD DAVID T. , GOLDSTEIN THEODORE P. , SELIGMAN ARNOLD M.
Published: April 2011

By synthesizing an analogue of N,N-dimethyl-p-phenylene diamine, a new reagent for the Nadi reaction was found which provided a useful histochemical method for either cytochrome oxidase (G-Nadi) or peroxidase (M-Nadi). The new agent is 4-amino-1-N,N-dimethylnaphthylamine and yields with α-naphthol, an indonaphthol purple pigment which is more stable and more satisfactorily distributed in tissue than the indophenol blue of the Nadi reaction. Thin sections may be used, some permanence of color is obtained and the granules are fine and anatomically distributed. The localization of cytochrome oxidase activity in heart, stomach, spleen, and cerebellum of the rat is described.
HISTOCHEMICAL DEMONSTRATION OF PHOSPHORYLASE IN BRAIN TISSUE: ASSOCIATION OF POSTMORTAL NEURON CHANGES WITH PHOSPHORYLASE ACTIVITY
Authors: FRIEDE REINHARD L.
Published: April 2011

The preparation of brain tissue for the histochemical demonstration of phosphorylase induces postmortal cell changes, such as pycnomorphic, chromophilic neurons and swollen, vacuolated, chromatin depleted neurons. The inconsistent phosphorylase reaction in grey matter is associated with the following changes. A positive phosphorylase reaction is found in pycnomorphic, chromophilic neurons, whereas the swollen, homogenized neurons exhibit negative phosphorylase reaction of the cell body but sometimes a definite pericellular phosphorylase activity. Irregularities in the phosphorylase reaction in the grey matter are therefore related to postmortal cell changes and conclusions on the normal distribution of the enzyme should be drawn with extreme care. There is indication that a positive phosphorylase reaction might result from an adaptive cell change occurring during the treatment of the specimens in vitro.
INFLUENCE OF CHEMICAL STRUCTURE ON THE RATE OF AZO COUPLING AND ITS SIGNIFICANCE IN HISTOCHEMICAL METHODOLOGY
Authors: NACHLAS MARVIN M. , GOLDSTEIN THEODORE P. , ROSENBLATT DAVID H. , KIRSCH MARVIN , SELIGMAN ARNOLD M.
Published: April 2011

Reliability of enzymatic localization in tissue sections by the simultaneous coupling techniques is dependent to a great extent upon the speed of coupling. Therefore, the influence on coupling rate of the structure of the diazonium ion and of the coupling component was studied. Electro-negative groups in the diazonium ion increase the rate of coupling, while the same groups in the coupling component decrease the rate. Electro-positive groups in the coupling component accelerate coupling, but slow it when present in the diazonium ion. The relation of coupling rate and electro-negativity of the substituents in the diazonium ion follows Hammett's equation (8). Although the relations is linear on coupling with aromatic hydroxy compounds, it is not so with aromatic amines. The most active diazonium ions showed no increase in coupling rate with aromatic amines. This suggests that for those enzymes hydrolyzing an ester link, increase of coupling rate might be accomplished by modifying the structure of either the coupling component used in the substrate or the diazonium salt. However, for enzymes splitting amide linkages, the only possibility of improving the localization is by modifying the structure of the coupling component.
A HISTOCHEMICAL STUDY OF INTRANUCLEAR INCLUSIONS IN MOUSE LIVER AND HEPATOMA
Authors: LEDUC ELIZABETH H. , WILSON J. WALTER
Published: April 2011

Intranuclear inclusions which develop in the hepatic cells of mice fed a methionine-rich basal/bentonite diet and inclusions which occur spontaneously in a transplantable hepatoma contain materials of cytoplasmic origin which have become segregated within the nucleus. These materials include sudanophilic lipids, glycogen, iron-containing pigments, and the enzymes acid and alkaline phosphatase, non-specific esterase and β-glucuronidase. In general, their presence in the inclusions varies with their abundance in the cytoplasm. Mitochondria and the succinic dehydrogenase activity associated with them have not been observed within these intranuclear inclusions. Ihe inclusions are always enveloped by a layer of desoxyribonucleic acid which resembles that which lines the nuclear membrane. They arise contiguous to the nuclear membrane and appear to increase in size gradually. Hence, it is possible that the inclusions may represent invaginations of the nuclear membrane in which cytoplasmic materials accumulate. It is also postulated, however, that inclusions could be and may be formed by accumulation of cytoplasmic materials which normally diffuse freely between the swollen chromosomal vesicles of the resting nucleus. The inclusions arise under a great variety of conditions, but they invariably occur in greatly enlarged nuclei. The altered relationship of nuclear surface to nuclear volume may play an important role in the formation of intranuclear inclusions.
A NEW TECHNIQUE FOR THE SIMULTANEOUS DEMONSTRATION OF LIPID AND ACID POLYSACCHARIDES ON THE SAME TISSUE SECTION
Authors: ZUGIBE FREDERICK T. , BROWN KENNETH D. , LAST JULES H.
Published: April 2011

A new combination staining technique which utilizes alcian blue for acid polysaccharides and oil red O in carbowax "400" for lipid is described. This technique, combined with a carbowax embedding procedre, provides a method which fulfills all of the requirements for the study of these substances in the same tissue section; i.e., (1) lipid and polysaccharide loss during the preparation of the tissue for staining is minimal, (2) the lipid stains sharply, (3) the polysaccharide stain is specific, (4) the stains contrast sharply, (5) the lipids and polysaccharides are identifiable "in statu nascendi", and (6) thin adjacent sections can routinely be prepared.
THE SEPARATION AND CHARACTERIZATION OF SUBMICROSCOPIC COMPONENTS OF HEPATIC CYTOPLASM
Authors: TAKANAMI MITURU
Published: April 2011

In an ultracentrifugal study on the cytoplasmic supernatant of rabbit liver, the following two principal components were separated from the supernatant by differential centrifugation and their biochemical properties investigated: (1) a granular substance sedimented at a rate of about 250s (250s component) and (2) a few macromolecular components the sedimentation rates of which were roughly in the range of from 40s to 100s (macromolecular components).

The 250s component, which was rich in lipids and easily disintegrated into smaller units by treatments of ultrasonic oscillation and of Nadesoxycholate, exhibited much higher activities of dipeptidase, acid and alkaline phosphatase than the macromolecular components. By contrast, the latter macromolecular components which belonged to ribonucleoprotein complexes exhibited comparatively high activities of RNase and esterase.

Uptake in vivo of radioactive phosphate (P32) by the RNA contained in the above two principal components markedly differed from each other. When the RNA contained in the 250s component was separated by the use of Nadesoxycholate into RNA in a non-sedimentable portion and that in a sedimentable portion corresponding to a ribonucleoprotein coplex, the RNA in the latter state showed an uptake rate extremely different from that of the macromolecular components. So it is emphasized that the ribonucleoprotein complex comprised in the 250s component and that existing free in the cytoplasm (i.e. macromolecular components) are metabolically different.
DETERMINATION OF SUCCINIC DEHYDROGENASE ACTIVITY IN TISSUE CULTURE
Authors: KAUFMAN NATHAN , HILL ROBERT
Published: April 2011

Two methods of demonstrating succinic dehydrogenase activity in tissue cultures of HeLa cells have been demonstrated. One method employs a protective layer of agar applied to the cells prior to incubation in the selected substrate. The other method uses a mixture of agar and substrate which is placed on the cells; incubation follows without the further addition of substrate.
DIFFERENTIATION OF NUCLEOPROTEINS BY INACTIVATION OF PROTEIN-BOUND AMINO GROUPS AND STAINING WITH TOLUIDINE BLUE AND AMMONIUM MOLYBDATE
Authors: LOVE ROBERT , LILES ROSCOE H.
Published: April 2011

1. A method of staining nucleic acids is described. The method depends upon (a) liberation of the phosphoryl groups of nucleic acids by inactivation of protein-bound amino groups; (b) binding of the phosphoryl groups by toluidine blue; and (c) coupling of some of the bound toluidine blue with polymolybdate. Nucleic acids are stained differently by toluidine blue-molybdate because of variation in the susceptibility of the amino groups of nucleoproteins to inactivation by formaldehyde and nitrous acid. At each stage of inactivation the amount of toluidine blue available to react with molybdate differs for each nucleoprotein. Polymolybdates react with toluidine blue to form a metachromatic complex involving the incorporation of water of dimerization. Hypochromia results when small amounts of this complex are formed at sites of orthochromatic staining by toluidine blue; larger amounts of this complex eclipse coloration by toluidine blue and produce intense metachromasia.

2. By means of the toluidine blue-molybdate stain the deoxyribonucleoprotein and 7 different forms of ribonucleoprotein can be distinguished in the cell. These are: the nucleolus, nucleolinus, parachromatin, perichromosomal, chromosomal, granular and diffuse cytoplasmic ribonucleic acid. The relationship of these observations to known chemical and functional differences in the nucleoproteins of the cell is discussed.

3. A study of seminiferous epithelium, embryonic, and neoplastic cells by the toluidine blue-molybdate method suggests that one type of ribonucleoprotein passes from nucleus to cytoplasm and vice versa during mitosis. Another form, the diffuse cytoplasmic ribonucleic acid, is unaltered during mitosis. It is suggested that these may correspond to the gene-dependent and gene-independent ribonucleic acid, respectively.
HISTOCHEMICAL STUDY OF INDUCED GASTROINTESTINAL TRACT TUMORS IN STRAIN A/JAX (LUNG TUMOR SUSCEPTIBLE) MICE
Authors: BURT ROBERT C. , KILLMEYER LOIS A. , GRAUER ROBERT C.
Published: April 2011

Tumors were induced in the small intestine, forestomach, and lungs of strain A/jax mice by feeding olive oil emulsions containing 20-methylcholanthrene.

Histochemical methods were used to determine the occurrence and distribution of acid and alkaline phosphatase in the tissues of the mice. The tumors generally showed a complete absence of alkaline phosphatase and widely varying quantities of acid phosphatase in the cytoplasm of the individual cells.

Tumors of the small intestine and forestomach showed a high concentration of alkaline phosphatase in the submucosal zone immediately beneath the neoplasm. No enzyme changes from normal were detected in lesions considered histologically to be premalignant.

Some groups of mice were fed iron choline citrate or ethylenediamine tetraacetic acid, tetrasodium salt in addition to 20-methylcholanthrene. These substances produced no effect on the rate of growth or numbers of tumors induced, nor on the acid and alkaline phosphatase reactions.