A nitrous acid procedure as a selective histochemical means of eliminating the N-sulphates of glycoconjugates

Summary A nitrous acid procedure has been shown to lead to the elimination of N-sulphates in sections of a series of tissues containing sulphated glycoconjugates. Two groups of sulphated glycoconjugate-containing tissues were used; one contained N-sulphates and other was devoid of such groupings. In the first group of tissues, mast cells of different origins and renal glomeruli in the rat were employed. Xiphoid and tracheal cartilage matrix, submandibular and sublingual gland acini and gastric, duodenal and colonic mucosae were used in the second group. Sections were treated with nitrous acid and then stained with Alcian Blue pH 1.0, high iron diamine or Aldehyde Fuchsin for sulphated glycoconjugates. Such treatment was found to diminish the staining intensities exclusively in N-sulphated glycoconjugate-containing structures such as mast cell granules and renal glomerular basement membrane, providing a means of chemically eliminating N-sulphates of glycoconjugates in tissues.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedure

Synopsis The high iron diamine (HID) method has been found to impart density at the ultrastructural level selectively to sites known to contain sulphated complex carbohydrates. Thus, immature primary granules in rabbit heterophils, immature précrystalloid granules in rabbit eosinophils, all granules of rabbit basophils, mouse and rat mast cells and the nucleoids of -granules of rabbit platelets were stained by HID. Granules of mast cells in rat cervical lymph node varied in the distribution pattern of the HID-reactive component. Mucous droplets within goblets of mouse colonic epithelial cells varied in HID reactivity. Sites known to contain sialomucin but no sulphates, such as mucous cells and apical plasmalemmae in mouse rectosigmoid colon, failed to stain with HID in contrast to their reactivity for dialysed iron at the ultrastructural level. The surface of mast cells and blood cells lacked affinity for HID, indicating that the dialysed iron binding at the surfaces can be attributed to neuraminic acid. HID proved more effective than dialysed iron in visualizing acid mucosubstance in precursor forms of the crystalloid granules in the eosinophil and in mast cell granules. Inclusion of 0.5% glycerol in the HID solution enhanced staining in mouse colon.     

Effects of strong electrolytes on the iron alum-Alcian Blue-Safranin staining of mast cell granules of the rat

Summary Rat mast cells fixed in Carnoy's fluid were stained with iron alum-Alcian Blue-Safranin solution after pre-treatment with strong electrolyte solutions including acids, neutral salts and alkalis. Although both red and blue mast cells were observed without pre-treatment, most mast cells were stained blue and a few red when they were stained after the pre-treatment. Mast cell granules contain salt complexes formed between basic proteins and acidic polysaccharides through ionic linkages between protein basic groups and polysaccharide sulphate and carboxylic acid groups. It is suggested that when sections are treated with strong electrolyte solutions, complexes are broken by disruption of ionic linkages and sulphate and carboxylic acid groups of polysaccharides masked by basic proteins become available for binding Alcian Blue. This was confirmed by model experiments performed with smears of a heparin-lysozyme complex.When mast cells were fixed in aldehyde-containing fixatives, no effects of strong electrolyte solutions on the staining properties of mast cell granules were revealed.     

Ultrastructural cytochemistry of glycoconjugates in basophils from humans and animals

The distribution of glycoconjugates in the basophil granules of humans, guinea pigs, and rabbits was compared. The observation of acid mucopolysaccharides using the dialyzed iron method and of sulfated glycoconjugates using the high iron diamine method revealed three types of reactions in the basophil granules of all three species: granules showing a strong overall reaction, granules showing reaction only at their periphery, and granules showing no reaction. With regard to the relationship between maturation and the types of basophil granules, it appeared that, in general, there were many type-1 granules among immature basophils, but that these granules decreased in mature basophils as type-3 granules increased. The reaction patterns of periodate-reactive neutral glycoconjugates, as shown by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, were different from those of acid mucopolysaccharides: the reaction of basophil granules was diffusely positive, and localization at the periphery was rarely observed. Therefore, unlike the acid mucopolysaccharides, it was difficult to classify the glycoconjugates into three types. However, as with acid mucopolysaccharides, there was a tendency for periodate-reactive glycoconjugates to decrease as maturation progressed. In terms of different species of animals, the reaction of periodate-reactive glycoconjugates with PA-TCH-SP was stronger in humans and rabbits than in guinea pigs.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Fluorescence of mast cell granules in paraffin sections and cell smears induced by an N-quaternary oxazole scintillator

Summary The N-quaternized derivative of dimethyl-POPOP (termed Q4) induces a bluish-green fluorescent reaction in mast cell granules from paraffin sections and cell smears, in addition to a previously described bluish-white fluorescent reaction in chromatin DNA. The chromatin reaction was abolished by staining the samples either with Mayer's Haematoxylin before Q4 treatment or by Q4 treatment at pH 1.5. The reaction in mast cell granules was absent after substrate methylation. The staining sequence Haematoxylin-Eosin-Q4 also worked well in paraffin sections, allowing the observation of the current histological image under bright-field illumination as well as double-colour emission under fluorescence microscopy. The sequence is proposed as a new diagnostic procedure for demonstrating mast cell granules.     

Ultrastructural and ultracytochemical identification of the small granules in basophils from human and animals

The small granules in the basophils obtained from humans and animals were compared ultrastructurally and cytochemically. Cytochemically, there were no qualitative differences among the small granules in the species examined. The small granules in humans, guinea pigs and rabbits were approximately 0.16-0.22 micron, 0.15-0.17 micron, and 0.12-0.16 micron, in diameter, respectively. In all species small granules had a single unit membrane and contained some amorphous material. In immature cells many of the small granules were distributed near the Golgi apparatus, while in the mature cells many of them were found around the periphery of the cell. There were no morphological or cytochemical differences between the small granules of the immature cells and those of the mature cells. The negative reaction in the dialysed iron and high iron diamine methods showed that the small granules did not have acid mucopolysaccharides or sulfated glycoconjugates. The strong reaction of the small granules of all species to the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) test, which was especially prominent in rabbit, showed that the small granules have many periodate-reactive neutral glycoconjugates but no acidic glycoconjugates. Enzyme cytochemistry revealed that the small granules are negative for peroxidase and catalase but positive for acid phosphatase.     

Ultrastructural cytochemistry of glycoconjugates in basophils from humans and animals

Summary The distribution of glycoconjugates in the basophil granules of humans, guinea pigs, and rabbits was compared. The observation of acid mucopolysaccharides using the dialyzed iron method and of sulfated glycoconjugates using the high iron diamine method revealed three types of reactions in the basophil granules of all three species: (1) granules showing a strong overall reaction, (2) granules showing reaction only at their periphery, and (3) granules showing no reaction. With regard to the relationship between maturation and the types of basophil granules, it appeared that, in general, there were many type-1 granules among immature basophils, but that these granules decreased in mature basophils as type-3 granules increased. The reaction patterns of periodate-reactive neutral glycoconjugates, as shown by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, were different from those of acid mucopolysaccharides: the reaction of basophil granules was diffusely positive, and localization at the periphery was rarely observed. Therefore, unlike the acid mucopolysaccharides, it was difficult to classify the glycoconjugates into three types. However, as with acid mucopolysaccharides, there was a tendency for periodate-reactive glycoconjugates to decrease as maturation progressed. In terms of different species of animals, the reaction of periodate-reactive glycoconjugates with PA-TCH-SP was stronger in humans and rabbits than in guinea pigs.     

Selective Staining of Mast Cell Granules with Chrysoidin

A simple chrysoidin stain (0.5% aqueous solution, 5-10 min) is selective for mast cell granules; alum-hematoxylin-chrysoidin stains histological features in addition to the granules and periodic acid-Schiff can be used for cytological and histochemical features. Selective chrysoidin staining is probably due to strong basophilia of mast cell granules.     

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

Mucosal mast cells of the gastrointestinal tract constitute a separate cell line within the mast cell system of the rat, differing in several respects from the classical connective tissue mast cells and, unlike the latter, requiring special fixation techniques for their demonstration. We have examined some histochemical properties of mucosal mast cells of the duodenum and compared them with connective tissue mast cells of the tongue or skin. The results indicate that the structural integrity of the granules of both types of mast cell is partly dependent on ionic linkages between glycosaminoglycan and protein. The so far unidentified glycosaminoglycan of mucosal mast cells appears to be more soluble than the heparin of connective tissue mast cells. The strongly fluorescent binding of Berberine to the granules of connective tissue mast cells and, depending on their content, of heparin is absent from mucosal mast cells, confirming previous findings which suggested that they contain a glycosaminoglycan with a lower degree of sulphation. Aldehyde fixation by routine procedures reversibly blocks the cationic dye binding of mucosal mast cell granules. The dye binding groups may be unmasked by trypsination or by long staining times of the order of several days. The results suggest that the blocking of staining by aldehydes is caused by a diffusion barrier of a protein nature. Mucosal and connective tissue mast cells thus differ with respect to the spatial arrangement of glycosaminoglycan and protein in their granules. As a result of the study a modified method for the demonstration of mucosal mast cells in tissue sections is described, based on normal formaldehyde fixation and staining in Toluidine Blue for a long time. It has some advantages over previous methods and preserves the structure of mucosal and connective tissue mast cells equally well.     

Keratan sulfate glycosaminoglycans in murine eosinophil-specific granules.

We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils. Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl alpha-2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules. Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules. High iron diamine-thiocarbohydrazide-silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids. Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD. These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II. (J Histochem Cytochem 47:481-488, 1999)     

Identification of a chymotrypsin-like proteinase in human mast cells

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.     

Immunofluorescent localization of an alkaline proteinase in skeletal muscles from diabetic rats

Alkaline proteinase (chymase) was localized in skeletal muscle tissues from seven day streptozotocin-diabetic rats. Extruded mast cell granules containing proteinase were visible in the extracellular space and inside certain myofibers from both extensor digitorum longus (EDL) and soleus muscles. Additional diffuse staining was present in the cytoplasm of many EDL fibers. This evidence provides support for a possible role of muscle cells in the endocytosis of mast cell granules.     

Histochemical Aspects of the Affinity of Mast Cell Granules for Night Blue

The affinity of mast cell granules for night blue was studied in fresh and fixed rat lip, dog mast cell tumor, normal human ileum, and human mast cell and carcinoid tumors. Fixatives used were 10% formalin, 1% trichloracetic acid in absolute alcohol, and Zenker's and Bouin's fluids. Extractions of fresh tissue with hot water, acids, and bases removed the stainable material or prevented staining, but similar treatment of fixed tissue did not. Hot pyridine was without effect as was chloroform-methanol, but methylation blocked mast cell staining by night blue. Chromic acid oxidation and prolonged Zenker and Bouin fixation also prevented staining. Hyaluronidase treatment was without effect. Sulfhydryl and disulfide linkages were changed without altering the stainability.     

Distribution, Histochemical and Enzyme Histochemical Characterization of Mast Cells in Dogs

This study describes the distribution, proteoglycan properties and protease activity of mast cells from 15 different dog organs. In beagles and mixed breed dogs, staining with Alcian Blue-Safranin O revealed mast cells in all the organs examined. However, their numbers varied and they demonstrated unique localization patterns within some of these organs. Berberine sulphate fluorescence-positive mast cells were observed in the submucosa, muscularis and serosa of the intestines, as well as the tongue and liver (within the connective tissue). Mast cells within the intestinal mucosa were negative for, or demonstrated weak, berberine sulphate staining. Heterogeneity of mast cells in terms of the proteoglycans contained within their granules was further confirmed by determination of critical electrolyte concentrations (CECs). The CECs of mast cells within the connective tissue of several organs, including the intestines (submucosal and muscularis-serosal layers) were all greater than 1.0 M. The results from CEC experiments together with berberine staining indicate that heparin was contained within their granules. Relative to the CECs of mast cells in other organs, mast cells in the intestinal mucosa exhibited lower CECs, suggesting that the proteoglycans within their granules were of lower charge density and/or molecular weight. Although mast cells were classified into two groups by proteoglycans within the granules, enzyme histochemical analysis in beagles revealed three subtypes of mast cells: chymase (MC(C)), tryptase (MC(T)) and dual positive (MC(TC)) cells. There was no correlation between the proteoglycan content and enzyme properties of the mast cell granules.     

鸡中枢淋巴器官肥大细胞的组织化学与形态学

对哺乳动物的,特别是啮齿动物和人类肥大细胞已有了比较深入的研究, 但关于家禽肥大细胞的研究很少.本研究旨在阐明鸡中枢淋巴器官中肥大细胞的组织化学与形态学特征.本研究证实Carnoy 氏液是鸡肥大细胞的优良的固定液,而中性缓冲福尔马林(NBF) 却阻断了大多数肥大细胞的着染力.甲苯胺蓝和阿尔新蓝是鸡肥大细胞的良好的染料,但阿尔新蓝能使更多的肥大细胞着染,虽然其也可使杯状细胞着染.作者的一种新的染色法, 长时间阿尔新蓝染色(LAB-S)可用于NBF固定的组织中肥大细胞的染色,因为其着染的细胞数与Carnoy 氏液固定甲苯胺蓝染色的细胞数无显著差异(P<0.001).在胸腺髓质中见有大量的肥大细胞,而胸腺皮质仅可见个别肥大细胞位于血管周围及小叶间结缔组织中.腔上囊的皮质与髓质中很少见有肥大细胞.肥大细胞有血管周围分布的倾向,但一个有趣的发现是血管内偶尔也有个别肥大细胞.电镜下可见肥大细胞的胞浆颗粒内充满无定形的颗粒状基质,但其电子密度有的较高,有的较低.少数胞浆颗粒内有旋涡状及网状亚微结构.但未见有人类肥大细胞胞浆颗粒内特征性的晶格状和卷轴状的亚微结构,也未见到在绵羊肥大细胞中描述过的特殊亚微结构.     

Conjugated avidin binds to mast cell granules

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.     

The use of a simple haematoxylin and eosin staining procedure to demonstrate micronuclei within rodent bone marrow

In the pharmaceutical industry, the majority of drug-safety evaluation studies are carried out preferentially in the rat. Consequently, drug absorption, distribution, metabolism and excretion profiles are available for this species. Such data usually have to be generated independently in the mouse, to allow validation of any micronucleus tests carried out in this species. Unfortunately, at the present time, the rat is not ideal for use in the micronucleus test due to the presence of large numbers of contaminating mast cell granules. Such granules are stained blue by the most commonly accepted staining procedure (May-Grunwald-Giemsa), and can be erroneously scored as micronuclei when they overlay erythrocytes. A simple haematoxylin and eosin staining procedure was evaluated in the micronucleus test using rats and mice. With this procedure, micronuclei stained blue-black and were readily distinguishable from cell inclusions resembling micronuclei such as mast cell granules, which remained unstained. Essentially similar quantitative data for micronucleus incidence and erythrocyte distribution were obtained in mice using this staining technique when compared to the use of the more established May-Grunwald-Giemsa staining procedure. However, unlike the use of the May-Grunwald-Giemsa procedure, the use of the haematoxylin and eosin stains allowed the accurate estimation of micronucleus incidence within the marrows of treated rats in the presence of contaminating mast-cell granules. Furthermore, unlike alternative procedures using fluorescent stains, the haematoxylin and eosin stained preparations are stable, constitute a permanent record of the experiment, and can be analysed at the convenience of the investigator. Therefore, this staining procedure may offer a useful alternative, for example, when evaluating rat bone-marrow smears within which considerable mast cell contamination can occur.     

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.