bluuebunny/crossref_metadata_2025 huggingface dataset:
Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver.
Authors: Maly I P , Sasse D
Published: April 2011

In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.
A tetramethylbenzidine/tungstate reaction for horseradish peroxidase histochemistry.
Authors: Weinberg R J , van Eyck S L
Published: April 2011

Tracing of neuroanatomical pathways commonly involves the histochemical demonstration of horseradish peroxidase, using the chromogen tetramethylbenzidine. A new modification of this reaction using ammonium paratungstate stabilizer retains high sensitivity while permitting the reaction to be performed at pH 6.0 in isotonic solutions. The reaction product resists solvents, allowing Nissl-stained sections to retain their peroxidase labeling. With subsequent stabilization by diaminobenzidine, the tissue is suitable for electron microscopic study and is compatible with post-embedding immunocytochemistry.
Immunohistochemical analysis of p53 and HER-2/neu proteins in human tumors.
Authors: Chang K , Ding I , Kern F G , Willingham M C
Published: April 2011

We examined samples of tumors of human breast, ovary, and colon of various degrees of malignancy for the expression of p53 protein, using a panel of anti-p53 antibodies and peroxidase immunohistochemistry. Of 66 tumor cases (24 cases of ovarian carcinoma, 23 cases of colon adenocarcinoma, and 19 cases of breast carcinoma), 36 (53%) showed high levels of expression of p53 using a human-specific antibody, and 16 (24%) showed high expression of a mutant form of p53. In the mutant p53-positive breast tumor samples, six (86%) were positive for HER-2/neu reactivity, compared with colon (0/4) and ovarian tumors (1/5). The pattern of p53 intracellular localization and tissue distribution, and the relationship between the expression of mutant p53 and cell differentiation, were also examined; poorly differentiated cells showed either overexpression of p53 or higher levels of mutant p53 in comparison with more normal cells.
Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.
Authors: van Lookeren Campagne M , Oestreicher A B , van der Krift T P , Gispen W H , Verkleij A J
Published: April 2011

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.
Exact ultrastructural localization of glutathione peroxidase in normal rat hepatocytes: advantages of microwave fixation.
Authors: Utsunomiya H , Komatsu N , Yoshimura S , Tsutsumi Y , Watanabe K
Published: April 2011

Glutathione peroxidase (GSH-PO), a highly soluble, selenium-dependent enzyme metabolizing lipid peroxides, is allegedly distributed in both the cytosol and mitochondria. With the pre-embedding method of immunoelectron microscopy for GSH-PO employing conventional immersion-fixation, the nuclei of rat hepatocytes stain positively, whereas mitochondria are negative. Such observations are inconsistent with the results of biochemical and immunoblot analyses using isolated subcellular fractions. In the present study, we employed the combination of microwave irradiation and fixation in 4% paraformaldehyde (PFA), with or without 0.1% glutaraldehyde (GA), to enhance the accuracy of ultrastructural localization of GSH-PO in rat liver. A small block of liver was irradiated by microwave for 10 sec in cold cacodylate-buffered 4% PFA containing 0.1% GA. After further immersion of the tissue in 4% PFA at 4 degrees C for 1-6 hr, the standard procedure for pre-embedding immunoelectron microscopy was employed. We observed partial inhibition of artifactual diffusion of cytosolic GSH-PO into the nuclei and consistent GSH-PO localization in mitochondria. Dual localization of this enzyme in the cytosol and mitochondria of normal rat hepatocytes was thus confirmed.
Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts.
Authors: Fukushima O , Yamamoto T , Gay C V
Published: April 2011

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.
Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand.
Authors: Lotti L V , Pavan A , Zompetta C , Mancini P , Faggioni A , Frati L , Torrisi M R
Published: April 2011

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.
Generation of lobuloalveolar development from isolated rat mammary ducts and end buds.
Authors: Rudland P S
Published: April 2011

Implantation of excised bud-free ductal fragments (DUCTS), terminal end buds (TEBs), or alveolar buds (ABs) from virgin mammary glands of Wistar-Furth rats into interscapular fat pads of syngeneic female rats produces, after 16 weeks, complete ductal outgrowths including TEBs and ABs. Treatment of the recipient rats with perphenazine for 1 day or mating them after 12 weeks and then isolating the resultant outgrowths after 16 weeks produces significantly larger outgrowths than those from untreated hosts. The outgrowths consist of distended ducts and lobules or distended ducts and alveoli, respectively. Histochemical and immunocytochemical staining of the outgrowths with reagents that depict epithelial, myoepithelial, and lactating alveolar cells (peanut lectin alone, monoclonal and polyclonal antibodies to rat caseins) indicate similar cell compositions and arrangements for all outgrowths irrespective of their source; these are also similar to the mammary glands of the perphenazine-stimulated or lactating hosts. There is one major difference: the degree of staining of peanut lectin alone and the anti-caseins is greater for outgrowths produced by the ABs and TEBs than for those produced by the DUCTs. DUCT implants left for 1 year after cessation of lactation of the hosts are still stained appreciably by peanut lectin alone and by the anti-caseins, particularly the luminal secretions. Therefore, the complete morphogenetic and cell differentiating ability for generating mammary glands is present in bud-free ducts, but this ability can be enhanced in TEBs/ABs or abnormally expressed at ectopic sites.
HISTOCHEMICAL DEMONSTRATION OF NORADRENALINE IN THE ADRENAL MEDULLA OF THE HAMSTER
Authors: ERÄNKÖ OLAVI
Published: April 2011

Formalin-induced fluorescence and a positive iodate reaction are both present exclusively in the peripheral adrenomedullary cells of the hamster. These cells contain principally noradrenaline.
PRESERVATION OF PHENYL-PHOSPHATASE ACTIVITY DURING PARAFFIN EMBEDDING
Authors: DALGAARD JÖRGEN B.
Published: April 2011

Model experiments with serum-gelatine pieces in which the effect of some basic histotechnical procedures on the phosphatase activity was studied, are reported. Following fixation and dehydration in alcohol, clearing in methyl-benzoate-celloidin, and embedding in paraffin at temperatures up to 53°C., the phosphomonoesterase activity was preserved, while embedding at higher temperatures caused increasing inactivation. The admixture of slight amounts of "soft paraffin" (m.p. 42°C.) to the ordinary 55° paraffin and embedding at 52-53° is advocated for phosphatase studies.
PROTEIN BINDING OF AZO DYES BY TISSUE HOMOGENATES
Authors: RUTENBURG ALEXANDER M. , SELIGMAN ARNOLD M.
Published: April 2011

In the colorimetric estimation of sulfatase activity in tissues based upon the conversion of 6-benzoyl-2-naphthol, liberated by enzymatic hydrolysis, to an azo dye by exposure to tetrazotized diorthoanisidine, a substantial fraction of the dye is adsorbed by tissue homogenate. This also occurs with protein fractions. The fraction of total dye adsorbed increases with protein or homogenate concentration and varies with the type of protein or homogenate used. The adsorption by protein conforms empirically to a general theoretic equation for the binding of azo dyes to protein. Azo dyes derived from 6-bromo- or 6-benzoyl-2-naphthol were adsorbed more extensively than the dyes derived from 1- or 2-naphthol.
A STRICTER INTERPRETATION OF THE FERRIC FERRICYANIDE REACTION WITH PARTICULAR REFERENCE TO THE DEMONSTRATION OF PROTEIN-BOUND SULPHYDRYL AND DI-SULPHIDE GROUPS
Authors: ADAMS C. W. M.
Published: April 2011

(1) Two distinct pigments (Prussian blue and Prussian green) are found in tissues after the ferric ferricyanide reaction; they have different chemical properties.

(2) Only strong reduction causes rapid formation of Prussian blue from ferric ferricyanide solutions. Conversely Prussian green may be formed by weak reduction or possibly by absorption of traces of this pigment present in ferric ferricyanide solutions. Brown or yellow pigments in tissues may appear green in the presence of low concentrations of Prussian blue. Thus no strict interpretation of green colours can be made.

(3) Sulphydryl groups, phenols, indoles, pyrroles, aromatic amines, uric acid and ascorbic acid reduced ferricyanide to Prussian blue. Certain oils, aliphatic amines, aldehydes and some inorganic reagents caused the production of Prussian green.

(4) Up to 5 minutes immersion in the ferric ferricyanide solution is recommended for the demonstration of sulphydryl groups in trichloracetic-alcohol fixed tissues.

(5) A technique is given for the demonstration of protein-bound disulphides, involving previous thioglycollate reduction followed by the demonstration of the formed sulphydryls by 1½ minutes immersion in the ferricyanide solution. This technique demonstrates protein containing at least 4 per cent of cystine (equivalent to 1 per cent of sulphur).
SOME ASPECTS OF THE MECHANISM OF ORCEIN STAINING
Authors: FULLMER HAROLD M. , LILLIE R. D.
Published: April 2011

Using the Taenzer-Unna acid orcein method of staining, it was demonstrated on formalin fixed tissues that orcein reactivity is not prevented by measures which block carboxyl, hydroxyl, amine or aldehyde groups when it combines with collacin and elastic tissue, and that collagen, ordinarily unreactive, reacts strongly when these polar groups are blocked.
STUDIES ON THE CONNECTIVE TISSUE OF THE COCK COMB: I. HISTOCHEMICAL OBSERVATIONS ON THE GROUND SUBSTANCE
Authors: SZIRMAI J. A.
Published: April 2011

1. The ground substance of the mucoid layer of the cock comb has been found to show a strongly metachromatic reaction with toluidine blue, but the PAS reaction was negative. The fibrils, which do not stain metachromatically, show a significant PAS positivity. The metachromatic ground substance is very easily soluble in water, while the PAS positive substance of the fibrils is more resistant to extraction. The metachromasia of the ground substance disappears below pH 3.0. No metachromasia could be observed on sections previously treated with gelatin solution.

In the capon comb the PAS positivity is restricted to the thick collagen bundles and no metachromatic ground substance is present.

2. The uptake of toluidine blue by the comb tissue was determined quantitatively. The values obtained with sections of the cock comb are about 15 µg/mg. wet tissue, decreasing to about 3 µg/mg. after extraction with dist. water. The capon comb bound about 2-3 µg toluidine blue per mg. wet tissue.

3. Hexosamine determinations on slices of the whole cock comb gave dry weight values of about 4%, while the mucoid layer alone yielded 1½ to 3 times higher values. After extraction with dist. water, the cock comb tissue loses about 75% of the total hexosamine. In the capon comb, a hexosamine content of about 0.5% was found; this value was not influenced by previous dist. water extraction.

4. The water content of the defeated cock comb was found to be about 89% of the dry weight; that of the isolated mucoid layer was even higher (90 and 94%). The water content of the capon comb is about 75%.
THE MECHANISM OF THE MARCHI TYPE OF METHODS FOR VISUALIZING DEGENERATING MYELIN. MODEL EXPERIMENTS WITH PURE COMPOUNDS
Authors: WOLMAN M.
Published: April 2011

The chemical changes occurring in myelin lipids do not explain the reason for the specificity of Marchi's stain for degenerating myelin.

Experiments were made with a number of pure compounds smeared on glass slides with the aim of finding the type of compound which might be responsible for the Marchi staining.

Among the compounds tested heparin was the only one which fulfilled the requirements. This finding suggests a working hypothesis for further work on the mechanism of the early changes during demyelinization.
THE REPLACEMENT OF THE ACID HYDROLYSIS OF THE FEULGEN REACTION BY BROMINATION
Authors: BARKA TIBOR
Published: April 2011

Acid hydrolysis can be replaced by bromination in the Feulgen reaction. Under the influence of bromination purine bases are released from deoxyribonucleic acid and potential aldehyde groups are revealed. The bromine-Feulgen reaction can be blocked with aldehyde reagents. Consideration of the mechanism of Feulgen reaction still present some unresolved questions.
CYTOCHEMICAL STUDIES OF MITOCHONDRIA IN LEPTOMONAD FORM OF LEISHMANIA DONOVANI, THE KALA-AZAR PARASITE
Authors: GUHA A. , PYNE C. K. , SEN B. B.
Published: April 2011

Supravital and other mitochondrial indicators have been used to study the mitochondria of the leptomonad form of L. donovani. A more or less regular pattern of existence of these bodies was noted which demonstrates the structural and physiological homogeneity in the mitochondrial reactions. The mitochondria are always found to be present in the anterior end from where the flagellum comes out.
HISTOCHEMICAL DEMONSTRATION OF AMINOPEPTIDASE
Authors: BURSTONE M. S. , FOLK J. E.
Published: April 2011

A histochemical method for demonstration of aminopeptidase in tissue sections using the substrates l-leucyl β-naphthylamide and dl-alanyl β-naphthylamide is presented. The method may also be quantitated on tissue sections.

The successful histochemical demonstration of the enzyme with both substrates is dependent upon the use of frozen-dried tissues.
HISTOCHEMICAL LOCALIZATION OF SPECIFIC OXIDATIVE ENZYMES: II. LOCALIZATION OF DIPHOSPHOPYRIDINE NUCLEOTIDE AND TRIPHOSPHOPYRIDINE NUCLEOTIDE DIAPHORASES AND THE SUCCINDEHYDROGENASE SYSTEM IN THE KIDNEY
Authors: STERNBERG WILLIAM H. , FARBER EMMANUEL , DUNLAP CHARLES E. , LOUVIERE CONCETTA M.
Published: April 2011

The histochemical distribution of the succinic dehydrogenase system, DPN diaphorase and TPN diaphorase has been studied in the rat kidney. The staining pattern for each enzyme shows a highly reproducible and distinctly different zonal distribution in the cortex and medulla and a corresponding regional distribution along the course of individual nephrons. With these methods, virtually all the different segments of the nephron can be sharply delineated and identified.

Collecting ducts in the papilla, unstained with the succinate method, are well stained with DPN and TPN diaphorase. TPN diaphorase gives good staining of the thin limbs of Henle which are so difficult to demonstrate by other methods.

With the succinate system and DPN diaphorase, the thin limbs of Henle are unstained, but the thick ascending limbs stain heavily. This accounts for the sharp demarcation in staining seems between the outer medullary zone and the papilla. It is interesting that, with the succinate system, the collecting ducts show an abrupt cessation of staining at the same level.

Vascular endothelium and glomeruli show delicate staining with both diaphorases but not with succinate. Similarly, smooth muscle and the epithelium of the renal pelvis are stained by the diaphorase methods, but poorly, if at all, with succinate.

A distinct inner non-glomerular zone of the cortex is present in the rat and mouse kidney and is composed principally of coiled terminal portions of proximal convoluted tubules. In these rodents, the terminal segment of the proximal convoluted tubule is not straight and no distinct medullary rays are present. The staining of the inner cortical zone is distinctively different by all three methods from the staining of the outer zone which contains the first portion of of the proximal convoluted tubule. This furnishes additional evidence for a functional division of labor in the proximal convoluted tubule.

In the papilla, fusiform cells of unknown function are present in parallel formations, oriented at right angles to the direction of the tubules. These are well demonstrated in the rat kidney by TPN diaphorase and are found to be even more abundant in a desert rodent, the kangaroo rat. Should these cells prove to have a contractile function, they might play a role in the regulation of urinary volume.

Studies of the chemical anatomy of the kidney using these enzyme methods offers further evidence that the nephron is divisible into more than the conventional number of functional segments.
HISTOCHEMICAL LOCALIZATION OF SPECIFIC OXIDATIVE ENZYMES III. EVALUATION STUDIES OF TETRAZOLIUM STAINING METHODS FOR DIPHOSPHOPYRIDINE NUCLEOTIDE DIAPHORASE, TRIPHOSPHOPYRIDINE NUCLEOTIDE DIAPHORASE AND THE SUCCINDEHYDROGENASE SYSTEM
Authors: FARBER EMMANUEL , STERNBERG WILLIAM H. , DUNLAP CHARLES E. , LOUVIERE CONCETTA M.
Published: April 2011

Histochemical stains are methods for the visual demonstration of the biochemical properties of organized tissues. They differ from "ordinary" tissue stains only in so far as they lend themselves to chemical interpretation and are reliable only to the extent that they accurately reflect the presence and localization of specific chemical substrates or activities. In this paper we have described the results of a number of experiments designed to test the reliability and usefulness of tetrazolium salts as histochemical indicators of specific oxidative enzyme activities.

Control studies dealing with substrate specificity and inhibitors gave further evidence of the specificity of tetrazolium staining methods for the succinic dehydrogenase system.

Studies on the possibility of diffusion and nonspecific deposition of reduced blue tetrazolium and neotetrazolium indicated that the blue reduction products (diformazans) of each do not deposit nonspecifically from the medium and once deposited are firmly fixed in the tissues. The red reduction products (monoformazans) may deposit nonspecifically from the medium. Hence their deposition and localization must be interpreted with caution. The red monoformazans of these two salts can readily be converted to blue diformazans in tissue sections by non-enzymatic reducing agents.

Blue tetrazolium in the unreduced form is reversibly bound in the cytoplasm of all cells of the rat kidney but not in the nuclei. In the staining process it is probable that tetrazolium, bound in the cytoplasm, is reduced to formazan irrespective of the continued presence of free tetrazolium in the medium. Since no binding of tetrazolium is demonstrable in any of the cell nuclei, the failure of nuclei to stain does not necessarily indicate the absence of enzyme. It was found that two other hydrogen acceptors, methylene blue and pyocyanin, are also bound by the cytoplasm of cells and when either of these compounds is added to tetrazolium staining media, tissue staining is greatly accelerated and enhanced.

Relative enzyme activities determined quantitatively in tissue taken from different anatomical regions of the rat kidney correspond fairly well with the intensity of histochemical staining.

Tetrazolium methods are now available for demonstrating the succinic dehydrogenase system, diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. These methods appear to satisfy most of the criteria which we have adopted for the specificity and reliability of histochemical stains. They yield preparations which are quite satisfactory for histochemical purposes. However, it has not as yet been demonstrated that the formazan deposits accurately indicate the intracellular localization of enzyme activity and hence cytochemical interpretations are open to question.
PARTITION OF ADENOSINE TRIPHOSPHATASE IN INTRACELLULAR FRACTIONS OF MATURE AND IMMATURE RAT CEREBRUM
Authors: JORDAN WILLIAM K. , MARCH RUTH
Published: April 2011

Experiments in which infant and adult rat cerebrum were fractionated by differential centrifugation afford evidence that differences exist between comparable fractions from immature and mature nervous tissue with respect to a number of properties. These include differences in layering characteristics, and in the partition of total nitrogen and of calcium and magnesium activated adenosine triphosphatase (ATPase) activities. The total nitrogen content and Ca and Mg ATPase contents of the supernatant fraction of infant cerebrum are strikingly higher than those of the comparable fraction from adult cerebrum. Also, the "specific activity" of ATPase in two of the larger cytoplasmic particle fractions from infant cerebrum is higher than in comparable adult fractions.
CARBOHYDRATE AND CARBOHYDRATE METABOLITE UTILIZATION BY ENZYME SYSTEMS OF MOUSE BRAIN AND LIVER MITOCHONDRIA
Authors: DU BUY H. G. , HESSELBACH M. L.
Published: April 2011

1. Both mouse brain and mouse liver preparations were found to utilize substrates characteristic of the following carbohydrate enzyme systems : the glycogen, glucose, fructose, mannose, fructose-1-phosphate (5), oxidative pentose phosphate (4), and tricarboxylic acid system.

2. Each of these enzyme systems was found on isolated brain mitochondria and a number on isolated liver mitochondria.

3. The implications of these findings in regard to the lability, normal enzyme system complement and functions of mitochondria are discussed.
ATTEMPTS AT HISTOCHEMICAL DEMONSTRATION OF ETHYLENE GROUPS, WITH PARTICULAR REFERENCE TO STAINING PROPERTIES OF HAIR CORTEX
Authors: LANDING BENJAMIN H. , HALL HAZEL E.
Published: April 2011

(1) The results of comparative staining of hair cortex, posterior pituitary, and several tissue lipids, with leukofuchsin, aldehyde-fuchsin, and toluidine blue, after a variety of pretreatments, are presented.

(2) The results confirm those of Lillie and Bangle, that induced Schiff-positivity of hair cortex is not due to oxidation products of cystine, while the metachromatic basophilia induced in hair cortex by appropriate reagents is so explained. However, whether the reactive group responsible for the Schiff-positivity is a part of keratin or of some other molecular entity has not been established.

(3) Preliminary results with four additional reagents capable of producing histochemically demonstrable products from ethylenic groups are presented. Selenium dioxide appears to have little promise for this purpose, and osmic acid potassium chlorate appears to oxidize both lipids and cystine. Iodoform peracetic acid has provided evidence of the occurrence of unsaturated entities, perhaps lipids, in hair cortex. Phosphorus pentachloride may have value as a reagent for demonstration of certain lipids by means of induced basophilia, but the mechansim of phosphate radical binding may well be esterification rather than substitution into ethylenic groups.

(4) Aldehyde-fuchsin appears to have particular affinity for certain reactive groups, of unknown nature but possibly neither carbonyl nor acid radicals, which are produced in sections by treatment with sulfuric acid permanganate.

(5) Phosphorus pentachloride, like phosphoryl chloride, leads to leukofuchsin-positivity of certain tissue entities. No evidence that this reaction is not due to carbonyl groups was obtained, but phosphorylating agents are of interest with regard to the chemical nature of reactive groups responsible for certain Schiff reactions.
THE EFFECT OF ASCORBIC ACID ON METACHROMASIA IN SOLUTIONS OF PROTEIN AND ACID POLYSACCHARIDE
Authors: UDUPA K. N. , DUNPHY J. ENGLEBERT
Published: April 2011

Under the conditions of these experiments the interaction between toluidine blue and heparin or chondroitin sulphate is affected quantitatively by the addition of albumin. This effect can be reversed by the addition of ascorbic acid to the solution. The mechanism of action of the ascorbic acid is a subject for further study.
STABILITY OF THE FEULGEN-DEOXYRIBONUCLEIC ACID ABSORPTION CURVE IN SITU WITH VARIATION IN NUCLEAR PROTEIN CONTENT AND OTHER FACTORS
Authors: KASTEN FREDERICK H.
Published: April 2011

1. The influence of certain variables upon the shape of the Feulgen absorption curve of mouse liver nuclei has been studied in situ using cytophotometric equipment. The accuracy and reproduciblity of the cytophotometer was ascertained by various procedures.

2. The absorption curve usually is represented by a primary peak at about 585 mµ and a slight secondary peak at about 550 mµ. Reduction or removal of nuclear protein by extraction or enzymatic procedures does not alter the shape of the curve.

3. Variations in hydrolysis times do not alter the shape of the curve obtained with nuclei fixed in Zenker's fluid. There are some differences indicated around the secondary peak of formalin-fixed tissue following different hydrolysis times.

4. Comparison of absorption curves of nuclei fixed in different fixatives shows negligible differences.

5. There are no differences in the absorption curves of nuclei isolated in one per cent citric acid and in nuclei isolated in 0.88 M sucrose.
A HISTOCHEMICAL INVESTIGATION OF NEUROSECRETORY SUBSTANCE IN THE RAT
Authors: HOWE A. , PEARSE A. G. E.
Published: April 2011

(1) The chemical nature of `neurosecretory substance' has been examined by a variety of histochemical methods in the rat.

(2) With the methods used it appears that this material is protein or polypeptide in character; the presence of lipid or carbohydrate is excluded, and it does not contain ribose nucleic acid.

(3) These findings are discussed in relation to the theory of neurosecretion and the source of posterior lobe hormones.
Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family.
Authors: Berg T , Wassdal I , Sletten K
Published: April 2011

The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.
Effects of vinblastine on calcium distribution pattern and Ca2+,Mg(2+)-adenosine triphosphatase in rat incisor maturation ameloblasts.
Authors: Eisenmann D R , Salama A H , Zaki A M
Published: April 2011

Vinblastine is known to affect secretory and transport functions of ameloblasts. The effects of vinblastine on distribution patterns of membrane-associated calcium and Ca2+,Mg(2+)-ATPase in maturation ameloblasts were investigated cytochemically. The potassium pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg(2+)-ATPase. Ultrastructural changes induced by vinblastine included dislocated organelles and reduction or elimination of the ruffled border of the ameloblasts. Membrane-associated calcium pyroantimonate deposits were markedly reduced. The intensity of Ca2+,Mg(2+)-ATPase reaction product was also markedly reduced by vinblastine. Concomitant reduction of membrane-associated calcium and Ca2+,Mg(2+)-ATPase lends support to a role for maturation ameloblasts in control of a cyclic pattern of influx of calcium to mineralizing enamel.
The effect of avidin-biotin interactions in detection systems for in situ hybridization.
Authors: Speel E J , Schutte B , Ramaekers F C , Hopman A H
Published: April 2011

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.
Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.
Authors: Fukui Y , Yamamoto A , Masaki R , Miyauchi K , Tashiro Y
Published: April 2011

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.
Autophagy, cathepsin L transport, and acidification in cultured rat fibroblasts.
Authors: Punnonen E L , Autio S , Marjomäki V S , Reunanen H
Published: April 2011

The mechanisms of enzyme delivery to and acidification of early autophagic vacuoles in cultured fibroblasts were elucidated by cryoimmunoelectron microscopic methods. The cation-independent mannose-6-phosphate receptor (MPR) was used as a marker of the pre-lysosomal compartment, and cathepsin L and an acidotropic amine (3-(2,4-dinitroanilino)-3'-amino-N-methyl-dipropylamine (DAMP), a cytochemical probe for low-pH organelles) as markers of both pre-lysosomal and lysosomal compartments. In addition, cationized ferritin was used as an endocytic marker. In ultrastructural double labeling experiments, the bulk of all the antigens was found in vesicles containing tightly packed membrane material. These vesicles also contained small amounts of endocytosed ferritin and probably correspond to the MPR-enriched pre-lysosomal compartment. Some immunolabeling was also visible in the trans-Golgi network. In addition, cathepsin L, DAMP, and large amounts of ferritin were found in smaller vesicles which can be classified as mature lysosomes. Early autophagic vacuoles were defined as vesicles containing recognizable cytoplasm. MPR, cathepsin L, and DAMP, but not ferritin, were detected in the early vacuoles. Inhibition of the acidification in the early vacuoles by monensin did not prevent the delivery of MPR and cathepsin L. The presence of MPR in the vacuoles suggests that cathepsin L is not delivered to early autophagic vacuoles solely by fusion with mature, MPR-deficient lysosomes. Furthermore, although lysosomes were loaded with endocytosed ferritin, it was not detected in autophagic vacuoles. Either the trans-Golgi network or the MPR-enriched pre-lysosomes may be the main source of enzymes and acidification machinery for the autophagic vacuoles in fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)
Detection of CD1 mRNA in Paneth cells of the mouse intestine by in situ hybridization.
Authors: Lacasse J , Martin L H
Published: April 2011

Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Although their function(s) remains unknown, it has been proposed that CD1 may present antigens to specific subsets of peripheral T-cells. We now provide evidence in support of this hypothesis through the demonstration by in situ hybridization that Paneth cells of the mouse intestine express CD1 mRNA. These cells are thought to be involved in the immunological regulation of intestinal flora and could accomplish this task through interactions with intestinal intraepithelial lymphocytes. The expression and localization of CD1 mRNA was confirmed by both autoradiographic and non-isotopic techniques. The relevance of these results to CD1 function as well as to Paneth cell biology is discussed.
Fast myosin light chain expression in chicken muscles studied by in situ hybridization.
Authors: Billeter R , Messerli M , Wey E , Puntschart A , Jostarndt K , Eppenberger H M , Perriard J C
Published: April 2011

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.
Histochemical carbonic anhydrase in rat inner medullary collecting duct.
Authors: Kleinman J G , Bain J L , Fritsche C , Riley D A
Published: April 2011

Rat inner medullary collecting duct (IMCD) secretes substantial amounts of H+. However, carbonic anhydrase (CA), a concomitant of H+ secretion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprising the IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportion have features of intercalated cells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third cell type, previously called "IMCD cells", appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these "IMCD cells" have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells clearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD.
Measurements of mitochondrial deficiencies in living cells by microspectrofluorometry.
Authors: Schneckenburger H , Gessler P , Pavenstädt-Grupp I
Published: April 2011

Microspectrofluorometric methods were developed for detection of mitochondrial metabolites and marker molecules in living cells. After excitation in the near UV and blue spectral ranges, respiratory-deficient strains of Saccharomyces cerevisiae showed higher levels of intrinsic fluorescence than corresponding wild types. This may be attributed to an increased emission by NADH and flavin molecules of the mutants. After incubation with the mitochondrial marker rhodamine 123, there was a strong indication that an energy transfer from flavin to rhodamine molecules occurred, which was more pronounced for the respiratory-deficient yeast strains. Skin fibroblasts obtained from patients with mitochondrial diseases showed approximately the same levels of autofluorescence and energy transfer but higher variances than a control cell line. These higher variances may result from a coexistence of intact and defective mitochondria.
Nonspecific binding of nucleic acid probes to Paneth cells in the gastrointestinal tract with in situ hybridization.
Authors: Garrett K L , Grounds M D , Beilharz M W
Published: April 2011

Nonspecific binding of a number of unrelated nucleic acid probes to cells in the crypts of Lieberkuhn was observed in the small intestine of mice with the in situ hybridization technique. Hybridization signal was localized to cells which, by virtue of their histological position, represented Paneth cells. This signal could not be removed by RNAse, DNAse, or proteinase K treatment, and was not removed after high-stringency washing conditions. This report indicates that caution must be exercised in the interpretation of in situ hybridization data when looking for nucleic acid sequences in the gastrointestinal tract.
Epitope-selected monospecific antibodies to recombinant antigens from Toxoplasma gondii reacted with dense granules of tachyzoites.
Authors: Matsuura T , Tegoshi T , Furuta-Matsuura M , Sugane K
Published: April 2011

Epitope-selected monospecific antibodies were applied to investigate the localization of antigenic molecules in Toxoplasma gondii by immunoelectron microscopy. Eighty cDNA clones encoding antigenic polypeptides were immunoscreened from lambda gt11 expression library with T. gondii infected mouse sera. Twenty different clones with no crossreactivity were selected from eighty clones. Monospecific antibodies to antigens derived from respective cDNA clones extracted from infected mouse sera by the epitope selection method were used in Western blot analysis and immunoelectron microscopy. Eleven antigens were detected with epitope-selected antibodies in lysates of T. gondii tachyzoites. Five of the antigens with molecular weights of 60, 40, 35, 28, and 27 KD were localized in the dense granules. Monospecific antibodies purified by the epitope selection method were useful for investigating the localization of antigens without preparation of a monoclonal antibody from a hybridoma.
Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.
Authors: van der Beek E M , Pool C W , van Eerdenburg F J , Sluiter A A , van der Donk H A , van den Hurk R , Wiegant V M
Published: April 2011

Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.