Histochemical reactions of gastrointestinal mucosubstances with orcein, high iron diamine and Alcian blue after prior oxidation of tissue sections.

The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections

Summary High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-HID and ox-HID) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive mucosubstances were found in the foveolar epithelium of the stomach and in goblet cells of small and large bowel.The study was supported by grants from Sigrid Juselius Foundation and Paulo Foundation, Helsinki, Finland     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections.

High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-Hid and ox-Hid) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive and in goblet cells of small and large bowel.     

Tunicate Endostyles: Histochemistry of Acidic Glycoproteins Re-examined in Ciona intestinalis and Styela plicata

Previous reports of tunicate endostyles have suggested that they contain little or no acidic glycoproteins in the glandular zones. The endostyles of Ciona intestinalis and Styela plicata were examined after anhydrous fixation with cyanuric chloride. Polyanions were stained with alcian blue (AB) at pH 2.5 or azure A, while sulfomucins were stained with high-iron diamine (HID) or AB at pH 1.0. Endostyles were also tested for sensitivity to acid hydrolysis (AH) and saponification. In Ciona zones 2 and 4 sometimes demonstrated positive HID and AB 1.0 responses. Almost invariably zone 6 was AB+ at pH 2.5; zones 2 and 4 were frequently responsive to AB, but less intense. Each of these 3 zones, when AB+, was sensitive to AH. Responses by zones 3 and 5 to AB (pH 2.5), azure A and saponification suggest that these zones contain mostly nuclear material. In secretory zones 2, 4 and 6 histological responses are consistent with the histochemistry of sialomucins. Zones 1 and 8 had sulfated material in the apical edges in both animal groups. Among the fixatives used for Ciona, only anhydrous fixation demonstrated most of the positive responses to polyanion-sensitive stains.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Histochemical and Biometric Study of the Gastrointestinal System of Hyla Orientalis (Bedriaga, 1890) (Anura,Hylidae)

This study was carried out to assess the localization of hyaluronic acid (HA) and the distribution of glycoproteins in the gastrointestinal system of adult Hyla orientalis. Histochemical analysis of the gastrointestinal system in H. orientalis showed that mucous content included glycogene and/or oxidable dioles [periodic acid/Schiff (PAS)+], neutral or acid-rich (PAS/AB pH 2.5+), sialic acid residues (KOH/PAS+) and acid sulphate [Aldehyde fuchsin (AF)+] glycoproteins. However the mucus content was not the same in stomach, small and large intestine. The mucus content of stomach included only glycogene and/or oxidable dioles and sialic acid residues. Besides these histochemical methods, the localization of HA was detected using biotinylated hyaluronic acid binding protein labeled with streptavidin-fluorescein isothiocyanate (FITC). In the extracellular matrix of the submucosa, the reaction for HA was evident. Since HA was located in submucosa beneath the epithelial layer of gastrointestinal system, it has a significant role in hydric balance, and essential to provide the gastrointestinal system integrity and functionality. According to biometric results, there were statistical differences between small and large intestine in terms of the amount of material stained positive with PAS/AB, PAS, KOH/PAS and AF/AB. Additionally, number of goblet cells in the small and large intestine was significantly different.Key words: Gastrointestinal system, goblet cell, glycoproteins, hyaluronic acid, amphibian, Hyla.     

结肠腺瘤的免疫组织化学及组织化学研究

本文应用免疫组织化学CEA、MC_5(PAP法)和组织化学AB(PH2.5)/PAS、HID/AB(PH2.5)染色方法,对43例结肠腺瘤、52例结肠腺瘤伴中、重度不典型增生,13例结肠腺瘤伴癌变,15例结肠腺癌和10例正常结肠组织进行了相关抗原及大肠粘蛋白的研究探讨。结果发现五类组织之间其着色含量都呈现一定的差异,尤其是随着腺瘤的异型程度增大,这种差异更为明显。本实验结果提示,上述染色法对结肠腺瘤癌变的逐渐演变过程的变化具有一定的参考价值。     

Histology and ultrastructure of the gut epithelium of the neotenic cave salamander, Proteus anguinus (Amphibia, Caudata)

Histological, histochemical, and ultrastructural features of the gut of the European endemic cave salamander Proteus anguinus were studied. The gut is a relatively undifferentiated muscular tube lined with a simple columnar epithelium containing numerous goblet cells. The mucosa and underlying lamina propria/submucosa are elevated into a number of high longitudinal folds projecting into the lumen. The enterocytes are covered apically with uniform microvilli. Irregularity in the arrangement of microvilli was observed. Occasionally, irregular protrusions of the cytoplasm appear between groups of microvilli. Pinocytotic activity occurs at the bases of the intermicrovillous space. Mitochondria are numerous in the apical cytoplasm and basally beneath the nuclei. The supranuclear cytoplasm contains most of the cell organelles. The lateral plasma membranes of adjacent cells interdigitate and are joined by junctional complexes. The periodic acid-Schiff (PAS) reaction, indicating neutral mucosubstances, is positive only in the apical brush border of enterocytes and in goblet cells. The goblet cells also stained with Alcian blue (AB), at pH 2.5, thus revealing the presence of carboxylated glycosaminoglycans. Compact aggregations of AB- and PAS-negative cells are situated directly below the epithelium. Mitotic figures are present in individual clusters of cells. The fine structure of cells in these clusters indicated that these cells could be responsible for renewal of intestinal epithelium. Numerous endocrine-like cells could also be seen. The closely packed smooth muscle cells and amorphous extracellular material with collagen fibrils constitute a net-like structure under the basal lamina that is very closely associated with the epithelium. There are numerous acidophilic granular cells between epithelial cells, in the lamina propria/submucosa, and between cells aggregations. They seem to be associated with nematode infections and possibly constitute a humoral defense mechanism.     

The fine structure of the stomach mucosa of the llama (Llama guanacoe)

Summary The epithelium of the fundic region mucosa of the hind stomach in the Llama guanacoe has been studied using morphological and histochemical methods. Morphology suggests that solute and water absorption may occur in the epithelium of the surface and of the foveolae, although this absorption can not be estimated because of the extensive secretion of the gastric glands. The same cells of the surface and foveolar epithelium show numerous secretory granules. The glands reveal neck cells, chief cells, a large number of oxyntic cells, four types of endocrine cells (A-like, ECL, D and EC), brush cells and wandering cells. PAS and Alcian blue reactions for light microscopy suggest a secretion of neutral and acidic mucosubstances in the surface and foveolar epithelium, of neutral mucosubstances only in the neck cells. Periodic acid-thiocarbohydrazide silver proteinate (PA-TCH-SP) reaction for electron microscopy confirms the presence of neutral mucosubstances within the secretory granules of the surface, foveolar and neck epithelial cells. In all these cells, the reaction product is also evident within sacculi and vesicles of the maturing surface of the Golgi apparatus. A positive PA-TCH-SP reaction also occurs on the membrane (and not on the contents) of the Golgi apparatus (maturing surface) and of the secretory granules of the chief cells as well as on the membrane of the Golgi apparatus and of apical vesicles and tubules of the oxyntic cells. In addition, silver granules slightly enhance the electron density of the contents of the secretory granules in the endocrine cells. Morphological and histochemical findings are discussed and compared with results described by others for monogastric mammals.     

Mucosubstance histochemistry of Brunner's glands, pyloric glands and duodenal goblet cells in the ferret

The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods. The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man. The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomucins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

Ultrastructural and histochemical study on gills and skin of the Senegal sole, Solea senegalensis

This study was undertaken to identify the normal ultrastructural features of gills and skin of the Senegal sole, Solea senegalensis, for a comparative measure to morphological alterations caused by environmental stressors such as reduced water quality and diseases. In the Senegal sole skin, four morphologically distinct layers were identified: cuticle, epidermis, dermis and hypodermis. The epidermis was composed of stratified epithelium containing three cellular layers: the outermost or mucosa layer, the middle or fusiform layer and the stratum germinativum or the basal layer. In the mucosa, two mucous cell types were differentiated: type A cells containing several round vesicles of different electron density and type B cells containing mucosomes of uniform electron density. Senegal sole have five pairs of gill arches, each containing two rows of well‐developed and compactly organized primary filaments and secondary lamellae. Fingerprint‐like microridges were observed on the surface of epithelial cells. The branchial lamellae epithelium consisted of different cell types: pavement, mucous and chloride. Between the chloride cells and the larger pavement cells, accessory cells were observed. Complexes of tight junctions and desmosomes were frequently observed between adjacent chloride and epithelial cells. Neutral mucosubstances and/or glycoconjugates were observed in the epidermis, dermis and hypodermis of S. senegalensis skin. Proteins rich in different amino acids, such as arginine and cysteine, reacted negatively or weakly positive in the epidermis, dermis and hypodermis. In gills, some mucous cells responded weakly positive to periodic acid‐Schiff (PAS) reaction but were strongly stained with Alcian Blue at pH 0.5, 1 and 2.5. When Alcian Blue pH 2.5–PAS reaction was performed, most mucous cells were stained blue (carboxylated mucins) and some mucocytes stained purple, indicating a combination of neutral and acid mucins. Proteins rich in cysteine‐bound sulphydryl (‐SH‐) and cystine disulphide (‐S‐S‐) groups were strongly detected in branchial and epidermal mucous cells, whereas lysine, tyrosine and arginine containing proteins showed very weak staining in both epidermal and branchial mucous cells. Protein reactions were strongly positive in the pillar cells, except for those rich in tryptophan, whereas the branchial cartilaginous tissue did not show an important reaction. The performed lipid reactions were negative in goblet and chloride cells. It is concluded from this study that ultrastructural and cytohistochemical features of the Senegal sole skin and gills may serve as control structures in both natural and aquaculture systems to monitor or detect environmental stress responses at the histological level.     

南方鲇消化道杯状细胞分布及类型探讨

本研究应用阿利新蓝－过碘酸雪夫氏（ＡＢ－ＰＡＳ）染色方法对南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ）消化道杯状细胞的分布特点进行了分类研究,结果表明：南方鲇消化道杯状细胞的数量从肠前段、肠中段到肠后段,呈现递增趋势,有杯状、囊状和梨形三种不同的形态,ＡＢ反应阳性,ＰＡＳ反应阴性,分泌物主要含酸性粘多糖,属Ⅱ型粘液细胞。     

Alcianblue/PAS or PAS/alcianblue? Remarks on a classical technic used in carbohydrate histochemistry

The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-positive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Alcianblue/PAS or PAS/alcianblue?

Summary The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-posltive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Histochemical investigations on the secretory cells in the oesophagogastric tract of the Eurasian green toad,Bufo viridis

The secretory cells of the oesophagogastric tract of the Eurasian toad, Bufo viridis, were examined using standard histochemical methods and lectin histochemistry. Two goblet cell types were found in the oesophageal epithelium, differing in their morphology and the histochemical features of the secretory granules. These contained mainly acidic glycoconjugates, both sulphated and carboxylated, and a small amount of pepsinogen. Type I goblet cells contained stable class-III mucosubstances, which were absent in Type II. No pluricellular oesophageal glands were found. The oesophagogastric junction had a superficial epithelium similar to that of the oesophageal epithelium, with alveolar pluricellular glands, secreting stable class-III mucins, and few oxynticopeptic cells. The gastric mucosa presented secretory cells both in the surface epithelium and in the gastric glands. Superficial and foveolar cells produced neutral mucins with Gal1,3GalNAc residues. Neck cells, oxynticopeptic cells and endocrine cells were found in the gastric glands. Neck cells produced stable class-III mucosubstances. A functional gradient was observed in the oxynticopeptic cells from the oral to the aboral fundus, with a decrease in pepsinogen secretion towards the aboral fundus and a possible increase in HCl secretion. In the pyloric mucosa, the oxynticopeptic cells disappeared and the glands produced only neutral mucins, without stable class-III mucosubstances.     

Some histochemical reactions of mast cells of gerbils,hogs, armadillos and cats

Summary Mast cells of the Mongolian gerbil Meriones unguiculatus, the hog Sus scrofa, the cat Felis catus and the armadillo Pasypus novemcinctus were studied histochemically in relation to various fixation procedures, using azure A at pH 1 and 3, alcian blue at pH 1 and 2.5, diazosafranin at pH 3 and 7.8–8, and the PAS reaction. Fixations were performed in buffered 10% formol and 5% glutaraldehyde, in Kose's fluid, buffered sublimate (B4), lead nitrate and lead acetate formol.With azure A and alcian blue many mast cells were found in the gerbil with the aldehyde fixatives, fewer with the heavy metals. The diazosafranin reaction was present only in the aldehyde material, the PAS reaction was negative.In the hog, mast cells were more numerous after heavy metal fixation, fewer with aldehydes. Azure A stained metachromatically at pH 1 and 3, alcian blue reacted only at pH 1, the PAS reaction was negative, the pH 3 and 8 diazosafranin reactions were positive with all 4 fixations.In the cat, mast cells were moderately numerous with lead acetate formol, rare with formol and absent with glutaraldehyde. They stained with azure A at pH 1 and 3, with alcian blue at pH 1 and 2.5, with diazosafranin at pH 3 and 8 and by the PAS reaction.Armadillo mast cells were more numerous after heavy metal fixations, stained with azure A and alcian blue at pH 1 and 2.5 to 3, and with acid and alkaline diazosafranin.The mast cells of the 4 species vary in their requirements for aldehyde and heavy metal fixation, in their PAS reactivity and in their pH 2.5 alcian blue staining. All are sufficiently sulfated to react to cationic dyes at pH 1, but vary in PAS reactivity, indicating partial or complete sulfation. The presence of 5-hydroxytryptamine is indicated in all four species.Assisted by grant from National Cancer Institute C-04816.     

The histochemical specificity of High Iron Diamine—Alcian Blue

Summary The specificity of the High Iron Diamine—Alcian Blue pH2.5 (HID—AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine—Alcian Blue pH 1.0 demonstrated that complete or almost complete staining ofO-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID—AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied transitional mucosa in human colonic diseases. Caution should be used in drawing conclusions from the use of HID—AB 2.5 without confirmatory evidence from other more specific procedures.     

Comparative histochemistry of the mucoproteic cells of the hypophysis from Rhamdia hilarii, Hypostomus punctatus, Prochilodus scrofa and Cyprinus carpio (Teleostei). Immunohistochemical identification of the gonadotropic cells

Adenohypophyseal cells showing positive histochemical reactions for mucosubstances were classified as type I-IV in Hypostomus (Plecostomus) punctatus (Loricariidae), Rhamdia hilarii (Pimelolidae), Prochilodus scrofa (Prochilodontidae) and Cyprinus carpio (Cyprinidae) according to cell shape, size, cytological characteristics and adenohypophyseal distribution. Cell types I and II are common to the four species, with each cell type showing very similar cytological and histochemical characteristics, in spite of different adenohypophyseal distribution of cell type II, according to the teleost species. Type I cells are globular basophils located in the proximal pars ditalis and are positive to PAS and Alcian blue pH 2.5 (AB) reactions, showing cytoplasmic vacuoles and changes in granule concentration in the mature phase of the gonadal cycle. The smaller type II cells are fusiform or oval basophils exhibiting a strong AB reaction but also reacting to PAS. Type III cells are located in the pars intermedia showing PAS-positive reaction. Considering different teleost species, these cells exhibit some variations specially in relation to cell size and shape which are not detected in mature male C. carpio. Otherwise cell type IV is only present in the rostral pars distalis of P. scrofa. They are weakly basophilic and negative to PAS, reacting strongly to AB. Only cell type I showed unequivocally positive immunohistochemical results with anti-salmon gonadotropin.     

小肠上皮分泌细胞特性的组织学观察

目的寻找一种可以替代人体消化管的动物标本,并通过特殊染色方法,使得小肠上皮分泌细胞的形态特征能够明显地显示出来。方法随机采集成年猫小肠的新鲜标本,经Bouin液灌注固定24h后,石蜡包埋切片脱蜡入水。分别采用Gomori染色法、PAS反应、Gomori＋PAS反应、阿利新蓝（alcian blue,AB）染色法、AB＋PAS反应、HE染色法和苏木精-焰红染色法进行染色。结果在各种染色的切片标本上,能够观察到杯状细胞的形态、分布和染色特性以及肠内分泌细胞的特点,并发现在它们之间还存在一种绿色颗粒细胞和嗜酸性颗粒细胞。结论通过特殊染色可以肯定猫的小肠杯状细胞合成的是中性粘蛋白和酸性粘蛋白;绿色颗粒细胞为未成熟杯状细胞;嗜酸性颗粒细胞为Paneth细胞,其特点是单个分散分布。肠内分泌细胞与周围其他上皮细胞的染色对比明显而容易识别。