ARTICLE |
Correspondence to: Ann M. Dvorak, Dept. of Pathology/East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215.
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Summary |
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We evaluated an enzyme affinitygold ultrastructural technique designed to identify RNA-rich structures, based on an RNasegold (RG) probe in human mast cells (HMCs). As expected, the RG technique labeled RNA-containing ribosomes and nucleoli in HMCs. The heparin-rich secretory granules in HMCs were also labeled. Extensive studies revealed that HMCs isolated from lung or skin and sustained in short-term cultures, derived de novo in growth factor-supplemented cord blood cell cultures, or present in vivo in multiple sites all shared this property. We performed a large number of controls designed to examine the HMC granule binding characteristics of gold alone, of irrelevant protein or enzymegold reagents, of the role of charge and enzyme activity after various enzyme digestions, after blocking with macromolecules, after exposure to inhibitors of RNase, of heparin, or to irrelevant enzyme inhibitors, including staining of macromolecule-containing test agar blocks and a variety of combined absorption and digestion experiments of the binding of RG to HMC granules. These studies established that the RG method detected heparin in this site in conventionally prepared, well-preserved electron microscopic samples. These findings demonstrate a new use for this enzyme affinitygold technique in mast cell biology, based on the known property of heparin as an inhibitor of RNase. (J Histochem Cytochem 46:695706, 1998)
Key Words: enzyme affinitygold technique, human mast cell, granules, RNA, heparin, ribosomes, electron microscopy
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Introduction |
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Postembedding enzyme affinitygold ultrastructural methods have been developed to localize specific substrates to cell organelles in samples that are optimally prepared for excellent electron microscopic imaging (
Human mast cell (HMC) granules store proteoglycans that have been shown primarily to consist of heparin (and smaller quantities of chondroitin sulfate E) (
We believed that these properties and the known affinity of heparin for RNase might prove useful in imaging heparin in optimally prepared electron microscopic samples. We chose to test this hypothesis in a unique subcellular site of heparin, the HMC granule. We selected this test structure because it contains a large amount of heparin and because it should not contain RNA, the known substrate for which the RG method was developed. We found that the RG method reliably labeled heparin in HMC granules with a degree of label specificity and visibility of ultrastructural detail equal to or surpassing existing ultrastructural methods that label mast cell granule heparin (
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Materials and Methods |
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Materials
Sodium citrate, tetrachloroauric [III] acid, polyethylene glycol, heparin, histamine; heparinase I from Flavobacterium heparinum EC 4.2.2.7, histaminase (diamine-oxidase) EC 1.4.3.6, heparin type III agarose beads, Sepharose beads; DNA type I, DNase from bovine pancreas EC 3.1.21.1, bovine serum albumin (BSA), chondroitin sulfate (CS), proteinase K EC 3.4.21.64, pronase E from Streptomyces griseus E.C. 3.4.24.31, poly-L-lysine (PLL), gamma globulin, polyvinyl sulfate (PVS), protamine, guanidine-HCl, and normal goat serum (NGS) were all from Sigma (St Louis, MO). RNase A (bovine pancreas) EC 3.1.27.5, and RNA were from Worthington Biochemical (Freehold, NJ). Polyuridine (poly U) and polyadenine (poly A) were from LKB Pharmacia (Piscataway, NJ). Difco Bacto Agar was from Difco (Detroit, MI). Hydrochloric acid (HCl) and sodium arsenate were from Fischer Scientific (Fairlawn, NJ). Cationized ferritin (CF) was from Miles (Elkhart, IN). Cationized colloidal gold was from Electron Microscopy Sciences (Fort Washington, PA). Human serum albumin (HSA) was from Accurate Chemical and Scientific (Westburg, NY).
Source of Human Mast Cells
In vivo and in vitro sources of HMCs included biopsy specimens of human skin, breast, and lung, isolated, purified human lung or skin mast cells [some samples were maintained in short-term (6-hr) tissue cultures], and HMCs that developed de novo in culture systems from their precursors in cord blood cells. Methods of obtaining these varied sources of HMC as biopsy samples, as isolated, purified, and short-term cultured lung or skin mast cells, and as newly emerging HMCs in culture systems have all been reported in detail (
Fixation
Small blocks of human tissue biopsy specimens were fixed by immersion in a mixture of 2% paraformaldehyde, 2.5% glutaraldehyde, pH 7.4, in 0.1 M sodium cacodylate buffer containing 0.025% calcium chloride, for either 2 hr at room temperature (RT) (lung, breast) or 5 hr at RT (skin). Isolated, purified human lung or skin mast cells and mast cells arising in cultures from cord blood cells were fixed in suspension in a mixture of 1% paraformaldehyde, 1.25% glutaraldehyde, pH 7.4, in 0.1 M sodium cacodylate buffer containing 0.025% calcium chloride, for 1 or 2 hr at RT.
Tissue Processing
Samples were washed in 0.1 M sodium cacodylate buffer, then either postfixed in 0.2 M Sym-collidine-buffered 1.33% OsO4, pH 7.4, in osmium potassium ferrocyanide-reduced osmium tetroxide [OPF method (
Preparation of EnzymeGold Complexes
Colloidal gold suspensions (
RNaseGold Staining Procedure
As reported (
Cytochemical Controls
Specificity controls [as described (
Cationized ferritin (CF) (0.5 ml in 10 ml Hanks' balanced salt solution) and cationized colloidal gold (poly-L-lysine bound to 10-nm gold) were used to stain HMCs directly in sections placed on grids. Some samples that were stained with either cationized ferritin or cationized gold en grid were then stained with RNasegold. Additional samples were studied in which either the sections on the grids or the enzymegold reagent were incubated with the following reagents either individually or in various combinations: poly-L-lysine 0.5 mg/ml, BSA 1 mg/ml, human serum albumin (HSA) 5%, normal goat serum (NGS) 5%, CS 3 mg/100 µl, DNA Type I 1 mg/ml, gamma globulin 1 mg/ml, polyvinyl sulfate 0.1 mg/ml, protamine 1 mg/ml, guanidine HCl 1 mg/ml, sodium arsenate 0.001 or 0.002 M, histamine 12 mg/ml, RNA 1 mg/ml, heparin 12 mg/ml. Finally, samples were stained en grid with RNasegold previously absorbed with heparinagarose beads or Sepharose beads, with or without additional exposure of the RNasegold to RNA or heparin or exposure of the heparinagarose or Sepharose beads to RNase before adding RNasegold to the beads.
Quantitation
Gold particles were counted on HMC granules of samples stained with multiple different preparations of RNasegold as well as a variety of controls, and were expressed as the density of gold particles/µm2 of granule area. Statistical evaluations were done by the MannWhitney, NewmanKeuls, or KruskalWallis test.
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Results |
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RNaseGold-labeled Subcellular Sites in Human Mast Cells
HMCs stained with RG revealed gold-labeled granules, nuclei, nucleoli, and ribosomes (Figure 1). These labeled sites were evident in HMCs of diverse origin (Table 1), including human lung mast cells (HLMCs) in vivo and after isolation, purification ex vivo, and short-term culture intervals in vitro (Figure 1) and human skin mast cells (HSMCs) in vivo and after isolation and purification ex vivo and development of HMCs in vitro from agranular precursors in human cord blood cells that were cultured with recombinant human stem cell factor (rhSCF) (
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HMCs have large, electron-dense secretory granules with a variety of ultrastructural patterns. In general, these include scrolls, particles, reticular threads, crystals, homogeneous electron-dense material, and mixtures of these patterns (
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Experiments to Determine the Basis of RG Staining of HMC Granules
Electron-dense Reagents En Grid.
Two reagents were examined: cationized ferritin (CF) (
Sample Preparations. When postfixation with OsO4 and staining with uranyl acetate en bloc were omitted from sample preparation of HMCs, the intensity of RG staining was diminished but exceeded background levels for the samples.
Physical Parameters Related to the RG Reagent.
The degree of HMC granule staining was related to the age of the RG reagent. In general, the RG staining level for granules up to 2 weeks after preparation of the enzymegold complex was intense but dropped extensively after this time. Flocculation of the RG reagent, with a color change from red to purple (
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Staining Parameters Affecting RG Labeling of HMC Granules. The optimal time for staining grids with RG was 1 hr and the optimal temperature was 37C. When very short staining times were used (e.g., 5 min), RG labeling of HMC granules was evident but diminished compared to standard RG labeling of HMC granules (Figure 3). Similarly, reductions in RG staining were noted when temperatures of 20C and 4C were used for a 1-hr staining interval (compared to RG staining of granules at 37C) (Figure 3). The density of RG/µm2 of HMC granules was determined over a pH staining range spanning 8.5 to 4.5 (Figure 3). In general, the optimal pH for RG staining of HMC granules was pH 7.5, a value used for comparative and standard purposes. Significant quantitative reductions in RG label/µm2 granule occurred as the staining pH was increased or decreased (Figure 3).
Effect of General Inhibitors, Blockers, and Enzyme Digestions on RG labeling of HMC Granules.
We performed experiments in which the effect on RG granule labeling of general inhibitors, blockers, and enzyme digestions (by incubating the samples en grid in the test reagent or incubating the RG reagent in solution with the test reagent) was assessed. In addition, a single reagent was tested before, with, or after RG staining of samples. Furthermore, combinatorial studies of multiple reagents were done together, in sequential and reverse-sequential order. In these experiments, labeling of HMC granules was blocked and reduced, respectively, with exposure to NGS or HSA, but exposure to gamma globulin (Figure 2H) or DNA (Figure 2I) did not block RG staining of HMC granules (Table 2). BSA placed en grid before RG staining quantitatively reduced HMC granule label (Table 3) (Figure 2S). When BSA and RG were incubated in solution before staining, a reduction (but not significantly so) in RG labeling of HMC granules occurred (Figure 2R) (Table 3). Blocking with CS by incubation in solution with RG before staining (Figure 2M) or en grid before staining with RG qualitatively reduced but did not significantly change the RG label of granules (Table 3). Blocking RG with PVS abrogated HMC granule RG staining altogether (Figure 2N; Table 2).
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Inhibition of RG staining of HMC granules occurred with exposure of the RG reagent to guanidine-HCl (Figure 2O) or protamine before en grid staining (Figure 2P; Table 3); no significant change in RG granule staining was seen with sodium arsenate (Figure 2Q; Table 3).
Digestion of samples en grid with pronase E, proteinase K, or HCl (Table 2 and Table 3) did not significantly reduce RG labeling of HMC granules.
Effect of Specific Blocking and Enzyme Digestions on RG Labeling of HMC Granules. Blocking of the RG reagent in solution with heparin before RG staining yielded a marked reduction in granule RG label (Figure 4C; Table 4), but large aggregates were often present. When the heparin was placed en grid before RG staining, no significant change in label density occurred (Figure 4D; Table 4). When the RG reagent was absorbed by passage over solid-phase heparin before en grid staining, a marked reduction in granule staining occurred (Figure 4I; Table 4) (and the heparinagarose turned red) compared to the retention of granule RG staining when RG was passed over Sepharose beads alone (Table 4) (the Sepharose beads remained white). Heparinase digestion of the grid before RG staining resulted in diminished granule staining (Figure 4E; Table 4), whereas incubation of heparin and heparinase together with the RG reagent before staining the grid abrogated the reduction of staining effected by heparinase (Table 4). Histamine blocking in solution resulted in decreased RG labeling of granules (Figure 4G; Table 4), whereas histamine en grid did not affect RG staining (Table 4). Digestion of the grid with histaminase before RG staining did not significantly reduce HMC granule labeling with RG (Figure 4H; Table 4).
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Blocking of the RG reagent in solution or by incubating the grid with RNA simultaneously, or before RG incubation, yielded variable results, from a reduction in granule RG staining that did not achieve statistical significance (Figure 4J; Table 4) to the presence of large aggregates on the grid. When RNA en grid preceded the RG staining, no change in label density occurred (data not shown), and aggregates were excessive and random in distribution. RNase digestion of the grid before RG staining sometimes resulted in diminished granule staining but sometimes increased it and generally produced large, random aggregates of RG staining [as did incubation of the RG reagent with RNase before staining the grid (Figure 4K)]. Combining digestions of grids with either proteinase or pronase before RNase digestion and RG staining markedly increased HMC granule label (Figure 4M). Digestion of fixed HLMCs with RNase before further processing for electron microscopy and followed by RG staining en grid resulted in no change in granule label (data not shown). Therefore, RNase digestions resulted in decreased or increased, positive as well as negative, staining in variable approaches of 10 experiments. Moreover, the presence of large aggregates impeded accurate quantitation (Figure 4K).
We next evaluated a number of combined blocking and digestion experiments (Table 4). When the RG reagent was combined in solution with heparin and RNA before staining, it resulted in a significant decrease of granule staining (Table 4) compared to standard RG staining. However, compared to heparin blocking in solution only (Table 4) or to RNA blocking in solution only (Table 4), significant differences were not seen. When RNA and RNase were incubated in solution for 60 min before a second 60-min incubation with the RG reagent, followed by en grid staining, random aggregates formed and the granule label was undiminished (Table 4).
In general, solid-phase heparin was most effective in absorbing the HMC granule labeling ability of the RG reagent. We therefore incorporated its use into several combination schemes to assess the RG reagent. When RG was passed over Sepharose beads before staining, the granule label was retained and the beads remained white (Table 4). Passage of the RG reagent over heparinagarose beads before staining yielded a marked reduction of granule label and the beads turned red (Figure 4I; Table 4). Combination in solution of RG and RNA for 1 hr, then passage over Sepharose beads and en grid staining, resulted in an insignificant reduction of gold label in granules and the beads remained white. Combination in solution of RG and RNA for 1 hr, then passage over heparinagarose beads and en grid staining, resulted in a significant reduction of gold granule label, and the beads were red. When RG and heparin were incubated for 60 min in solution, passaged over Sepharose beads, and grids stained, the granule gold label was significantly reduced and the beads remained white. A double absorption with heparin, by first incubating RG and heparin together for 60 min, followed by passage over heparinagarose and en grid staining, resulted in the lowest density of granule gold label in the heparin absorption experiments, and the beads were stained red. We also evaluated the initial passage of RNase over either Sepharose or heparinagarose, preceding the passage of RG over each type of beads before en grid staining. In this case, granule RG label after Sepharose was significantly reduced (Figure 4N) but not after passing over heparinagarose (Figure 4O; Table 4).
RG Staining of Agar Blocks Containing Heparin, RNA, or Histamine. Standard reagent-containing agar blocks were prepared, fixed, and processed for electron microscopy identically to the osmium collidine uranyl en bloc schedule used for the majority of cell and tissue samples. Thin sections containing these test materials were stained en grid with the RG reagent. Agar blocks containing heparin or RNA stained with the RG reagent. Agar alone, Epon alone, and agar blocks containing chondroitin sulfate or histamine did not bind the RG reagent.
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Discussion |
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We provide an examination of the properties of an enzyme affinitygold ultrastructural technique designed to detect RNA-rich structures (
In HMCs of wide provenance, immature and mature secretory granules known to contain heparin (
The findings that we present are supported by extensive controls and quantitation, which define the use of this enzyme affinity technique to image heparin in human mast cells. Heparin has been used to inhibit RNase in biochemical experiments for years (
The controls that were done in our work demonstrated that HCM granules were not labeled with gold alone, with an irrelevant proteingold (BSAgold), or with an irrelevant enzymegold (DNasegold). An active enzyme was necessary for a positive binding reaction to HMC granules, as indicated by physical parameters that facilitated granule staining, suggesting that the binding to granules may not rely on charge alone. Charge was investigated further by directly visualizing two cationic electron-dense probes (cationized ferritin, cationic gold) bound to HMC granules that blocked subsequent RG staining. The cationic macromolecule PLL (used to render the gold cationic) alone also blocked RG granule staining, implicating charge in the binding reaction.
A series of enzyme or acidic digestions of samples en grid or of the RG reagent were done. These showed specificity for heparin, because digestion of the sample with heparinase [an enzyme specific for heparin (
A series of macromolecules was used to block RG staining of human mast cell granules. Statistically significant reduction of granule label with RG was present with heparin and polyvinyl sulfate, both known to be polyanionic competitive inhibitors of RNase (
Several inhibitors of RNase or heparin were examined. These showed that protamine [a heparin inhibitor that binds to heparin (
Histamine, a cation and a major component of HMC granules (
Test blocks were prepared in agar and processed identically to the electron microscopic samples (
We sought to determine whether the RG method was detecting RNA in HMC granules as well as heparin. Some of the earliest subcellular fractionation studies of another secretory granule, the pancreatic zymogen granule, reported small amounts of RNA in purified fractions (
A number of RNase digestion experiments were done to further address the issue of RNA in HMC granules. In general, digestion of the samples on grids (or exposure of RG to RNase) resulted in the production of aggregates of gold that were poorly quantifiable. In addition, their qualitative interpretation was often so variable as to be unhelpful. When RNase was passed over Sepharose before the RG reagent was similarly passed over Sepharose, a significant reduction in granule staining occurred, compared to the same procedure using heparinagarose. This, of course, could only be interpreted to mean that either heparin or both heparin and RNA are present in granules. This finding does not discriminate between these two possibilities, because RNase successfully competed with RG binding to heparin (or RNA) in the granule in the first case. In the second case, unlabeled RNase binding to the heparin-agarose abrogated this competition, allowing the RG reagent to stain heparin and/or RNA in granules. Altogether, these findings document the imaging of HMC granule heparin by bound RNasegold. Other methods, including subcellular fractionation studies, will be necessary to determine whether RNA is also present in granules.
In summary, we demonstrate a new use for the ultrastructural enzyme affinitygold method originally designed to detect RNA, i.e., to detect heparin in human mast cell granules. We also confirm that this method, as reported (
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Acknowledgments |
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Supported by PHS grant AI-33372.
We thank Peter K. Gardner for editorial assistance in the preparation of the manuscript.
Received for publication November 5, 1997; accepted January 22, 1998.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson NG, Wilbur KM (1951) Studies on isolated cell components. II. The release of a nuclear gel by heparin. J Gen Physiol 34:647-656[Medline]
Bendayan M (1981) Ultrastructural localization of nucleic acids by the use of enzyme-gold complexes. J Histochem Cytochem 29:531-541[Abstract]
Bendayan M (1989) The enzyme-gold cytochemical approach: a review. Hayat MA, ed. Colloidal Gold. Principles, Methods, and Applications. Vol 2. San Diego, Academic Press, 117147
Brooks EM, Binnington KC (1989) Gold labeling with wheat germ agglutinin and RNAse on osmicated tissue embedded in epoxy resin. J Histochem Cytochem 37:1557-1561[Abstract]
Bussolati G, Gugliotta P (1983) Nonspecific staining of mast cells by avidin-biotin-peroxidase complexes (ABC). J Histochem Cytochem 31:1419-1421[Abstract]
Carlson SS, Wight TN (1987) Nerve terminal anchorage protein 1 (TAP-1) is a chondroitin sulfate proteoglycan: biochemical and electron microscopic characterization. J Cell Biol 105:3075-3086[Abstract]
Cheniclet C, Bendayan M (1990) Comparative pyrimidine- and purine-specific RNAsegold labeling on pancreatic acinar cells and isolated hepatocytes. J Histochem Cytochem 38:551-562[Abstract]
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299[Medline]
Craig SS, Irani A-MA, Metcalfe DD, Schwartz LB (1993) Ultrastructural localization of heparin to human mast cells of the MCTC and MCT types by labeling with antithrombin III-gold. Lab Invest 69:552-561[Medline]
Danon D, Goldstein L, Marikovsky Y, Skutelsky E (1972) Use of cationized ferritin as a label of negative charges on cell surfaces. J Ultrastruct Res 38:500-510[Medline]
Dvorak AM (1987) MonographProcedural guide to specimen handling for the ultrastructural pathology service laboratory. J Electron Microsc Tech 6:255-301
Dvorak AM (1991) Biochemical contents of granules and lipid bodiestwo distinctive organelles found in basophils and mast cells. In Dvorak AM, ed. Basophil and Mast Cell Degranulation and Recovery. Vol 4. In Harris JR (ser. ed.). Blood Cell Biochemistry. New York, Plenum Press, 2765
Dvorak AM (1992) Human mast cells. Ultrastructural observations of in situ, ex vivo, and in vitro sites, sources, and systems. In Kaliner MA, Metcalfe DD, eds. The Mast Cell in Health and Disease. Vol 62. In Lenfant C (ser. ed.). Lung Biology in Health and Disease. New York, Marcel Dekker, 190
Dvorak AM, Furitsu T, KissellRainville S, Ishizaka T (1992) Ultrastructural identification of human mast cells resembling skin mast cells stimulated to develop in long-term human cord blood mononuclear cells cultured with 3T3 mouse skin fibroblasts. J Leukocyte Biol 51:557-569[Abstract]
Dvorak AM, Mitsui H, Ishizaka T (1993a) Human and murine recombinant c-kit ligands support the development of human mast cells from umbilical cord blood cells: ultrastructural identification. Int Arch Allergy Immunol 101:247-253[Medline]
Dvorak AM, Morgan ES, Schleimer RP, Lichtenstein LM (1993b) Diamine oxidasegold labels histamine in human mast-cell granules: a new enzyme-affinity ultrastructural method. J Histochem Cytochem 41:787-800
Dvorak AM, Massey W, Warner J, Kissell S, KageySobotka A, Lichtenstein LM (1991) IgE-mediated anaphylactic degranulation of isolated human skin mast cells. Blood 77:569-578[Abstract]
Dvorak AM, Schleimer RP, Schulman ES, Lichtenstein LM (1986) Human mast cells use conservation and condensation mechanisms during recovery from degranulation. In vitro studies with mast cells purified from human lungs. Lab Invest 54:663-678[Medline]
Dvorak AM, Schulman ES, Peters SP, MacGlashan DW, Jr, Newball HH, Schleimer RP, Lichtenstein LM (1985) Immunoglobulin E-mediated degranulation of isolated human lung mast cells. Lab Invest 53:45-56[Medline]
Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature [Phys Sci] 241:20-22
Furitsu T, Saito H, Dvorak AM, Schwartz LB, Irani A-MA, Burdick JF, Ishizaka K, Ishizaka T (1989) Development of human mast cells in vitro. Proc Natl Acad Sci USA 86:10039-10043[Abstract]
Hay ED (1981) Extracellular matrix. J Cell Biol 91:205s-223s
Horisberger M (1979) Evaluation of colloidal gold as a cytochemical marker for transmission and scanning electron microscopy. Biol Cell 36:253-258
Jaques LB (1980) Heparinsanionic polyelectrolyte drugs. Pharmacol Rev 31:99-166[Medline]
Jorpes JE, Holmgren H, Wilander O (1937) Über das Vorkommen von Heparin in den Gefässwänden und in den Augen. Z Mikrosk Anat Forsch 42:279-300
Kobayashi Y (1962) Histamine binding by heparin. Arch Biochem Biophys 96:20-27[Medline]
Lagunoff D (1974) Analysis of dye binding sites in mast cell granules. Biochemistry 13:3982-3986[Medline]
Lagunoff D, Phillips M, Benditt EP (1961) The histochemical demonstration of histamine in mast cells. J Histochem Cytochem 9:534-541[Medline]
Lindahl U, Höök M (1978) Glycosaminoglycans and their binding to biological macromolecules. Annu Rev Biochem 47:385-417[Medline]
Linker A, Hovingh P (1972) Heparinase and heparitinase from flavobacteria. Methods Enzymol 28:902-911
Login GR, Galli SJ, Dvorak AM (1992) Immunocytochemical localization of histamine in secretory granules of rat peritoneal mast cells with conventional or rapid microwave fixation and an ultrastructural post-embedding immunogold technique. J Histochem Cytochem 40:1247-1256
McLaren KM, Pepper DS (1983) The immunoelectronmicroscopic localization of human platelet factor 4 in tissue mast cells. Histochem J 15:795-800[Medline]
Meldolesi J, Jamieson JD, Palade GE (1971) Composition of cellular membranes in the pancreas of the guinea pig. I. Isolation of membrane fractions. J Cell Biol 49:109-129
Mendelsohn SL, Young DA (1978) Inhibition of ribonuclease. Efficacy of sodium dodecyl sulfate, diethyl pyrocarbonate, proteinase K and heparin using a sensitive ribonuclease assay. Biochim Biophys Acta 519:461-473[Medline]
Oliani SM, Freymüller E, Takahashi HK, Straus AH (1997) Immunocytochemical localization of heparin in secretory granules of rat peritoneal mast cells using a monoclonal anti-heparin antibody (ST-1). J Histochem Cytochem 45:231-235
Paff GH, Bloom F, Reilly C (1947) The morphology and behavior of neoplastic mast cells cultivated in vitro. J Exp Med 86:117-124
Paff GH, Sugiura HT, Bocher CA, Roth JS (1952) The probable mechanism of heparin inhibition of mitosis. Anat Rec 114:499-505[Medline]
Rapraeger AC, Bernfield M (1983) Heparan sulfate proteoglycans from mouse mammary epithelial cells. A putative membrane proteoglycan associates quantitatively with lipid vesicles. J Biol Chem 258:3632-3636
Riley JF, West GB (1953) The presence of histamine in tissue mast cells. J Physiol 120:528-537[Medline]
Rossmann P, Matousovic K, Horacek V (1982) Protamine-heparin aggregates. Their fine structure, histochemistry, and renal deposition. Virchows Arch [B] 40:81-98[Medline]
Salmivirta M, Lidholt K, Lindahl U (1996) Heparan sulfate: a piece of information. FASEB J 10:1270-1279
Sanyal RK, West GB (1956) Binding of histamine in mammalian tissues. Nature 170:1293
Schick BP, Eras J (1995) Proteoglycans partially co-purify with RNA in TRI Reagent and can be transferred to filters by Northern blotting. BioTechniques 18:574-578[Medline]
Schulman ES, MacGlashan DW, Jr, Peters SP, Schleimer RP, Newball HH, Lichtenstein LM (1982) Human lung mast cells: purification and characterization. J Immunol 129:2662-2667
Skutelsky E, Roth J (1986) Cationic colloidal golda new probe for the detection of anionic cell surface sites by electron microscopy. J Histochem Cytochem 34:693-696[Abstract]
Skutelsky E, Shoichetman T, Hammel I (1995) An histochemical approach to characterization of anionic constituents in mast cell secretory granules. Histochem Cell Biol 104:453-458[Medline]
Stevens RL, Fox CC, Lichtenstein LM, Austen KF (1988) Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells. Proc Natl Acad Sci USA 85:2284-2287[Abstract]
Taper HS (1979) Evaluation of the validity of the histochemical lead nitrate technique for alkaline and acid deoxyribonuclease. J Histochem Cytochem 27:1483-1490[Abstract]
Taylor S, Folkman J (1982) Protamine is an inhibitor of angiogenesis. Nature 297:307-312[Medline]
Tharp MD, Seelig LL, Jr, Tigelaar RE, Bergstresser PR (1985) Conjugated avidin binds to mast cell granules. J Histochem Cytochem 33:27-32[Abstract]
Thompson HL, Schulman ES, Metcalfe DD (1988) Identification of chondroitin sulfate E in human lung mast cells. J Immunol 140:2708-2713
Uvnäs B, Åborg C-H (1976) An in vitro-formed protamine-heparin complex as a model for a two-compartment store for biogenic amines. Acta Physiol Scand 96:512-525[Medline]
Uvnäs B, Åborg C-H, Bergendorff A (1970) Storage of histamine in mast cells. Evidence for an ionic binding of histamine to protein carboxyls in the granule heparin-protein complex. Acta Physiol Scand 78(suppl 336):1-26[Medline]
Vogel KG, Peterson DW (1981) Extracellular, surface, and intracellular proteoglycans produced by human embryo lung fibroblasts in culture (IMR-90). J Biol Chem 256:13235-13242
Wingren U, Enerbäck L (1983) Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties, with special reference to protein blocking and solubility of the granular glycosaminoglycan. Histochem J 15:571-582[Medline]