Diffusion Artifacts Research Articles

Fine structural localization of glucose-6-phosphatase activity in the pancreatic islets of two amphibian species ( Salamandra salamandra L. and Rana esculenta L.)

The ultrastructural distribution of glucose-6-phosphatase activity has been investigated in the salamander and frog pancreas. In the pancreas of salamander the enzyme was located in the A-cells, while in frog it occurred in all main types of islet cells B-, A-, and D-cells). As a rule, the reaction intensity was higher in the frog islet cells. No reaction was recorded in the exocrine pancreatic tissue of both species. The glucose-6-phosphatase activity was constantly detected in the lumen of rough endoplasmic reticulum and between the nuclear envelopes. Other enzyme localizations, observed especially in the A-cells of the salamander pancreas, were considered possible diffusion artifacts ro remnants of other phosphatase activity. The enzyme distribution in different types of islet cells, as well as its functional significance are discussed in relation to the findings of other authors.

Diffusion artifacts in dating by stepwise thermal release of rare gases

Age plateaux and isochrons in the 40Ar- 39Ar and similar dating techniques can be severely altered by processes changing the geometric distribution of one isotope relative to the other. Age plateaux and isochrons can even be generated entirely as experimental artifacts. Alterations of 40Ar- 39Ar plateau ages by recoil redistribution of 39Ar, incorporation of trapped 40Ar and prior 40Ar loss provide significant examples. 10% shifts in isotopic ratios are very easily obtained and would result in errors in 40Ar- 39Ar plateau ages of 4 AE old samples of ∼100 m.y., which is comparable to the age differences which must be resolved to develop early lunar and solar system chronology. The possible occurrence of diffusion artifacts must be evaluated in every case to establish that ages and age differences obtained by stepwise thermal release analyses are real. All studies involving the stepwise thermal extraction of multiple isotopic components may show similar diffusion artifacts. Constant isotopic compositions may be obtained during thermal release which do not represent the actual compositions of sample reservoirs.

Iodide, thiocyanate and cyanide ions as capturing reagents in one-step copper-thiocholine method for cytochemical localization of cholinesterase activity.

The necessity of the presence of iodide in Cu-ThCh reaction was investigated by following the precipitate formation "in vitro" and by evaluating the ultrastructural localization of the precipitate in sympathetic ganglion cells of the frog and in the end-plate regions of the rat diaphragm. It was found that thiocyanate or cyanide is the only anion that can be substituted for iodide as the capturing agent in precipitation. The optimal concentration in the preincubation and incubation media of any one of the three anions is from 2 to 5 mM. At a concentration below 1 mM precipitation "in vitro" is considerably delayed as a result of which in electron microscopy diffusion artefacts appear in tissue sections. The unconverted primary precipitate obtained in the presence of iodide had been used for ultrastructural localization of ChE activity and now this use has been extended to precipitates obtained in the presence of CN- or CNS-. Better-quality localization in the presence of either one of the latter anions suggests that they, and particularly CN-, should be substituted for I- in the one-step Cu-ThCh method for the cytochemistry of cholinesterases.

Mechanisms of lysosomal enzyme release from leukocytes. IV. Interaction of monosodium urate crystals with dogfish and human leukocytes.

In order to determine the possible mechanisms whereby interactions between phagocytic cells and crystals of monosodium urate (MSU) lead to cell death with simultaneous release of both cytoplasmic and lysosomal enzymes, phagocytic leukocytes of the smooth dogfish shart Mustelus canis were studied by means of light and electron microscopy, and biochemistry. Lysosomes of these cells can be stained supravitally with toluidine blue and are large enough (0.7-0.8 mu) to be clearly resolved with the light microscope. Light microscopic observations showed that of cells exposed to MSU 87% of those containing visible ingested crystals died within 1 hour, whereas 92% of adjacent cells in the same wet mount without such srystals survived. Cell death occured after a latent period of 10-15 minutes following fusion of lysosomes with crystal-containing phagosomes. Electron microscopic examination of both dogfish and human leukocytes exposed to MSU for more than 1 hour and then fixed in situ revealed occasional discontinuities or ruptures in secondary lysosome membranes. Endogenous peroxidase activity could be cytochemically localized in primary and secondary lysosomes and in the cytoplasm adjacent to such ruptured secondary lysosomes. It was not seen adjacent to primary lysosomes, a result indicating that the cytoplasmic reaction product was not a diffusion artifact. To exclude the possibility that crystals were exercising their affect primarily upon the plasma membrane, suspensions of dogfish buffy coat cells were incubated with cytochalasin B (5 mug/ml, 10 minutes), which inhibits phagocytosis but not exocytosis of lysosomal enzymes by stimulated phagocytes. Whereas cells exposed to MSU crystals released 30% of their content of lysosomal beta-glucuronidase activity and 28% of their cytoplasmic lactate dehydrogenase (LDH) activity within 3 hours, preincubation with cytochalasin B reduced the release of LDH activity within that period to 6% but reduced the release of beta-glucuronidase activity only to 20%. Preincubation with 10-3 M cyclic adenosine monophosphate (cAMP) and theophylline (10-3 M), which inhibit lysosomal fusion, reduced the release of both LDH and beta-glucuronidase activities to 7% and 6% respectively. Cells that were preincubated with both cytochalasin B and cAMP + theophylline released only 1% LDH activity and 4% beta-blucuronidase activity. These results are compatible with the "suicide sac" hypothesis of lysosomal enzyme release mediated by MSU for the following reasons: a) cell death was seen to follow uptake, not mere exposure to crystals, b) ultrastructural studies indicated that the primary injury was to the secondary lysosome membrane, and c) cell death was reduced when either phagocytosis or lysosomal fusion was inhibited.

Localization of androgen target cells in the rat testis: autoradiographic studies.

Testosterone and 5α-reduced androgens maintain spermatogenesis in hypophysectomized rats (1,2). A specific receptor protein for testosterone and dihydrotestosterone has been demonstrated in testis (3–6), however, the androgen target cells containing these receptors have not yet been identified. In the present study, we used an autoradiographic technique (7), which avoids translocation and diffusion artifacts during tissue preparation. Experiments were carried out to determine whether the accumulation of androgen can be localized to specific types of cells within the testis.

The chemistry of the silver precipitation method used for the histochemical localization of ascorbic acid.

The usefulness of reductive silver precipitation for the histo chemical localization of assorbic acid in mouse lung has been examined under standard conditions. The estimated stoichiometry of the reaction shows that only one quarter of the silver precipitated in fresh tissue by the silver nitrate reagent was due to ascorbic acid. Although no evidence was found that diffusion of ascorbic acid proceeded more rapidly than reductive silver precipitation in the tissue, diffusion artifacts at the cellular led would seem likely to occur. Thiosulphate was shown to be more effective than ammonia m removing un reacted silver from tissues treated with silver nitrate.

Diffusion artifacts in cytochemistry of catalase.

The various causes of diffusion artifacts in cytochemical localization of catalase in rat hepatic peroxisomes have been investigated. It is noted that storage of tissue in buffer after the fixation with glutaraldehyde is mainly responsible for diffusion of catalase from peroxisomes. In the vicinity of such peroxisomes with diffusion, the product of oxidation of 3,3'-diaminobenzidine tetrahydrochloride (DAB) is localized on ribosomes and membranes of mitochondria and endoplasmic reticulum. In contrast, when the same tissue is reacted for catalase immediately after the fixation, no evidence of diffusion is observed. Prolonged incubation (up to 4 hr at 37°C) and storage of tissue after the reaction with DAB (up to 9 months) do not cause any diffusion of the reaction product. Similarly, there is no evidence of diffusion around the peroxisomes when the complete incubation medium is perfused into the liver immediately after the fixation with glutaraldehyde (perfusion-incubation technique). These findings indicate that the diffusion of heme protein catalase, rather than the diffusion of oxidation product of DAB, is responsible for focal cytoplasmic staining, which is seen occasionally in the vicinity of some peroxisomes.

A new method for the investigation of the kinetics of the capture reaction in phosphatase cytochemistry. I. Theoretical aspects of the local formation of crystalline precipitates.

Factors governing the precipitation kinetics and localization of lead phosphate in phosphatase cytochemistry are discussed, with special attention to the origin of diffusion artifacts. The principal differences between the kinetics of coupling reactions leading to insoluble organic dyes and of lead salt methods are treated in terms of the physicochemical theory of crystallization of sparingly soluble salts. This process, since it involves nucleation and crystal growth, is dependent on supersaturation and the presence of heterogeneous catalysts in the reaction medium. Under cytochemical conditions in tissue sections these factors are partially determined by the lead concentration in the medium and the local specific enzyme activity. It is postulated that heterogeneous nucleation of phosphate ions which have escaped from their enzymatic production sites may be a source of falsely localized precipitates. The need for experimental models for the study of the dynamics of precipitation and diffusion is stressed.

A histochemical study of the oxidation of 17β-estradiol in human term placenta at different incubation conditions

The present study deals with the histochemical demonstration of 17β-estradiol dehydrogenase in human term placenta using the polyvinyl alcohol method to reduce diffusion artefacts. Incubations took place with both NAD+ and NADP+ as coenzymes and at different pH values of the incubation medium. The NAD+ linked enzyme reaction showed a greater activity than the NADP+ linked, both in the trophoblast as well as in connective tissue. There were differences in staining intensity at the different pH values, and strongest reaction was observed using glycine-NaOH buffer pH 10 in the incubation medium. Owing to a non-enzymatically reduction of nitro blue tetrazolium by reduced NAD+, the demonstration of 17β-estradiol dehydrogenase is independent of diaphorase at this high pH. The findings are discussed in relation to data about “nothing dehydrogenase” and biochemically determined pH optima for the enzymatic reactions dealt with in this work.

Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology.

Three enzymes in the catecholamine synthesis—dopa decarboxylase (DDC), dopamine-β-oxidase (DBH) and phenylethanolamine-N-methyltransferase (PNMT)—have been isolated. Subsequently antibodies to these enzymes were prepared and used in immunohistochemical studies mainly with the aim to elucidate methodological problems. The indirect immunofluorescent technique was used throughout the study. It was found that cryostat sectioning followed by fixation in acetone or alcohol, a standard procedure in immunohistochemistry, was successful only with antibodies to the granule bound enzyme DBH. With antibodies to DDC and PNMT, two cytoplasmic enzymes, on the other hand, the results were hampered by diffusion artefacts. These drawbacks could be prevented by a brief aldehyde fixation, preferably by perfusion before cryostat sectioning. The best results were obtained with formalin followed by hydroxyadipaldehyde and acrolein. However, after glutaraldehyde fixation no specific fluorescence at all was observed. Freezing of fresh adrenals, followed by freeze-drying, treatment with formaldehyde vapours and paraffin embedding was tested but consistent results were only obtained with antibodies to PNMT. A new instrument, the Vibratome®, which allows sectioning of unembedded fixed or unfixed tissue, was used and successful results were obtained with all three antibodies. Furthermore, the possibility with this instrument to combine the immunohistochemical technique e.g. with the formaldehyde fluorescence method for visualization of monoamines is demonstrated. It is emphasized that the Vibratome ® technique may be a valuable tool for immunohistochemical studies on the central nervous system.

The effect of polyvinyl alcohol and polyvinyl pyrrolidone on diffusion artifacts in lactate dehydrogenase histochemistry.

The effect of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP), alone and in combination, on diffusion artifacts in histochemical incubations has been investigated using LDH as model enzyme. By measuring the amount of formazan in the medium at the end of the incubation it has been shown that both substances, but especially PVA, are effective in limiting diffusion. The significance of this is discussed in general as well as in relation to other procedures used to reduce diffusion artifacts.

Problems in the Measurement of Tissue Respiration with the Oxygen Electrode

Tissue respiration measurement with membrane- covered oxygen electrode, discussing effects of electrode deterioration and diffusion artifacts on accuracy

Peroxidase activity in rat liver microbodies after amino-triazole inhibition.

The in vivo effects of 3-amino-1,2,4-triazole (AT) on the fine structure of microbodies in hepatic cells of male rats has been studied by the peroxidase-staining technique. Within 1 hr of intraperitoneal injection AT abolishes microbody peroxidase-staining, and the return of staining coincides temporally with the known pattern of return of catalase activity following AT inhibition; this is further evidence that the peroxidase staining of microbodies is due to catalase activity. Peroxidase staining reappears in the microbody matrix without evidence of either massive degradation or rapid proliferation of the organelles. Furthermore, during the period of return of activity, ribosomal staining occurs adjacent to microbodies whose matrix shows little or no peroxidase staining. These observations are interpreted as evidence that (a) catalase is capable of entering preexisting microbodies without traversing the cisternae of the rough endoplasmic reticulum or the Golgi apparatus, and that (b) the ribosomal staining is probably not cytochemical diffusion artifact and may represent a localized site of synthesis or activation of catalase.

Nucleolar orthophosphate ions. Electron microscope and diffraction studies.

Lead acetate (3-10%, pH between 4.3 and 7.0, alone or containing 2% glutaraldehyde), when used as fixative, has been demonstrated to produce an intracellular microcrystalline precipitate of lead orthophosphate, Pb(5)(PO(4))(3)OH (lead hydroxyapatite). This confirms earlier work with the light microscope (6). In interphase cells the nucleoli are sharply delimited by the massive lead phosphate precipitate. Some diffuse precipitate is found in the nucleoplasm; it is always delimited by the nuclear membrane. Nucleolar localization of this orthophosphate pool is not a diffusion artifact; the pool is probably in a loosely bound state and is not retained by conventional fixatives. In maize root cells in advanced mitotic stages the lead phosphate crystals are seen distributed throughout the cytoplasm and also relatively concentrated on the late anaphase-early telophase chromosomes. This pool of inorganic phosphate anions may be involved in the mitotic cycle of chromatin condensation, and it may be partially responsible for the absence of mature ribosomes in the nucleolus through the chelation of divalent cations. It is evident that the siver-reducing component detected in the nucleoli of fixed cells (6) is a completely different substance.

New diazo reagent for specific staining of conjugated bilirubin in tissue sections.

A new technique is presented for the specific histochemical demonstration of conjugated bilirubin in tissue sections, using the diazonium salt of ethylanthranilate. The specificity of this method was proved by using test materials and cholestatic tissue sections. There was no diffusion artifact under the prescribed working conditions. The method is applicable to fresh cryostat sections and frozen sections of cold, formol-calcium-fixed tissues. The method could not be satisfactorily applied for the staining of total bilirubin. The method using 2,4-dichloraniline remains the best available technique for the histochemical demonstration of total bilirubin, while the technique with ethylanthranilate proves to be the most specific method available for the demonstration of conjugated bile pigments.

Mast cell stain for histamine in freeze-dried embedded tissue.

A new method is described which permits staining of the histamine in mast cells of embedded tissue. Small specimens of fresh tissue, rapidly frozen in isopentane-liquid nitrogen, are dried in vacuo at –35°C, embedded in paraffin, sectioned and stained with o-phthalaldehyde in ethyl benzene. Mast cell histamine appears bright yellow as viewed under fluorescence microscopy. The procedure is standardized, suitable for a wide variety of tissues and remarkably free of diffusion artifacts. The uncut blocks may be stored for months with no changes in later histamine-staining potential. Alternatively, embedding may be done in polyethylene glycols with satisfactory results.

Methods for the study of small phagosomes and their relationship to lysosomes with horseradish peroxidase as a "marker protein".

Small phagosomes (micropinocytic vesicles and vacuoles) which had taken up injected horseradish peroxidase were identified by staining for peroxidase with benzidine and H2O2. Because of the small size of the granules and the possibility of artifacts, previously described procedures had to be modified in several respects. Prefixation of the tissue by perfusion at 37°C prevented artifacts of diffusion and adsorption of peroxidase. The blue product of the reaction of peroxidase with benzidine in the small phagosomes was preserved and fading to brown was prevented by cooling the tissue section to –10° to –15°C during its processing through polar media. The blue reaction product was stable as soon as the section was transferred to an apolar medium. Small phagosomes were visualized together with lysosomes and phago-lysosomes in the same cells by double staining for acid phosphatase and peroxidase in contrasting colors. The incubation for acid phosphatase was performed at 4°C since low temperature increased the stability of peroxidase in the acid medium. Factors which form the basis for other improvements of the procedure are discussed.

ATP hydrolysis by isolated embryonic heart nuclei using a histochemical method.

SummaryA histochemical method was modified to demonstrate ATP hydrolysis by nuclei isolated from embryonic myocardium. Under a variety of experimental conditions, the results are in agreement with previous histochemical studies using cryostat sections and with enzymatic studies using isolated nuclei. The data give further support to the localization of ATP phosphohydrolase activity both at the nuclear surface and in the nucleo-plasm, under conditions which eliminate possible diffusion artifacts resulting from enzymatic activity by other cell components and non-enzymatic hydrolysis of ATP by Pb++.The author is indebted to Mrs. A. P. Breland and Mrs. A. T. Klein for technical assistance and to the University Medical Center Illustration Department for photography.

The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope.

Abstract The thiocholine technique for cholinesterase has been successfully adapted for the demonstration of enzyme distribution with the electron microscope. After fixation in buffered glutaraldehyde, thin slices of tissue were taken through a histochemical procedure designed to minimize diffusion artifacts and cytological damage; appropriate areas were dissected out, fixed with osmium tetroxide and embedded in Araldite. This technique has been used to study the distribution of enzyme in and around known cholinergic neurons in the rat. In diaphragm muscle the intense staining of the motor end plates is confined to the actual synaptic clefts. The distribution of cytoplasmic staining was similar in all three types of cholinergic neurons studied (ventral horn cells from the cervical cord, cells from the dorsal motor nucleus of the vagus, and cells from the hypoglossal nucleus). There was intense staining of the space contained within the individual bilaminar sheets of the rough endoplasmic reticulum and occasionally in areas of the nuclear envelope. Mitochondria, lysosomes, the smooth endoplasmic reticulum and most of the plasma membrane were unstained. In cholinergic nerve fibres staining was particularly intense at the axonal membrane and absent from the myelin sheath. In several regions of the brain known to receive an afferent cholinergic innervation many of the identifiable synaptic areas were heavily stained. Staining here was usually present round most of the presynaptic process, spreading over part of the postsynaptic process and often penetrating into the actual synaptic space. The mitochondria, microvesicles and the general cytoplasm of the synaptic processes were all unstained. In some areas, such as the hippocampus, there was intense membrane staining of fine cholinergic neuropil. The enzyme specificity of the technique is not in doubt and evidence is adduced for the view that diffusion artifacts are small. The close correlation between the electron-microscopic results and evidence from physiological and biochemical investigations is discussed.

Nucleoside phosphatase and cholinesterase activities in dorsal root ganglia and peripheral nerve.

IN DORSAL ROOT GANGLIA AND PERIPHERAL NERVE OF THE RAT AND OTHER SPECIES, NUCLEOSIDE PHOSPHATASE AND UNSPECIFIC CHOLINESTERASE REACTION PRODUCTS ARE FOUND IN THE PLASMA MEMBRANES AND SPACES BETWEEN THEM AT TWO SITES: (1) Schwann cell-axon interfaces and mesaxons of unmyelinated fibers, and (2) sheath cell-perikaryon interfaces and interfaces between adjacent sheath cells. Acetylcholinesterase reaction product is found in the perikaryon (within the endoplasmic reticulum) and the axon (axoplasmic surface). Nucleoside phosphatase reaction product is also found in the numerous vacuoles at the surface of perineurium cells, ganglion sheath cells, and cells surrounding some ganglion blood vessels. Nucleoside phosphatase activities in the sections fail to respond, in the manner described for "transport ATPase," to diisopropylphosphofluoridate, sodium and potassium ions, and ouabain. Nucleoside diphosphates are hydrolyzed more slowly than triphosphates in unmyelinated fibers, and are not hydrolyzed at the perikaryon surface. Nucleoside monophosphates are either not hydrolyzed or hydrolyzed very slowly. In contrast to these localizations, which are believed to demonstrate sites of enzyme activity, it is considered likely that diffusion artifacts account for the nucleoside phosphatase reaction product frequently found along the outer surfaces of myelinated fibers and within vacuoles at the Schwann cell surfaces of these fibers. The diffuse reaction product seen in basement membranes of ganglion and nerve may also be artifact.