An interesting case of colloidal iron positivity

Summary An interesting case of a colloidal iron (CI) positive basophilic substance in the adrenal medullary cells of amphibia and reptilia is reported here. The substance, however, does not stain by alcian blue (AB). It is negative to PAS, Azure A, aldehyde fuchsin, AB at pH 1 and MgCl₂ — AB though orthochromatically stained by toluidine blue at pH 3. More work is needed to establish the exact nature of the CI positive material.     

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.     

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.     

Staining Tomato Fruit Cuticle and Exocarp Tissues

Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue 8GX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylinalcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at × 100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues magenta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.     

Mucopolysaccharides in the dog spinal cord and dorsal root ganglia. A histochemical study.

A histochemical study of mucopolysaccharides in the dog spinal cord and dorsal root ganglia is reported in this paper. The histochemical techniques used were the following: PAS, colloidal iron, toluidine blue (pH 5.4 and 3.5), thionine (pH 5.4 and 3.5) and alcian blue 8GX (pH 1 and 2.5). Some histological stains were used also. Two types of neurons could be observed in spinal cord sections stained with colloidal iron techniques. In some neuron a border line of mucosubstances could be seen. In the dorsal root ganglia, different patterns of Nissl bodies distribution in neurons were described. This different distribution of Nissl bodies is associated with different metachromatic colorations of neurons. By using the colloidal iron method, two types of neurons were also revealed in the dorsal root ganglia: some neurons are of a yellow, small-sized and star-shaped type and others are of a light green, larger and round-shaped type. Mucosubstances in the endoneurium and perineurium of nerve fibers, in the Ranvier nodules and in the Schmidt-Lantermann incisures were observed. The possibility that the functional rhythm in some cases might be responsible for the difference in coloration between the dorsal root ganglia neurons is suggested.     

A postembedding staining method of intensifying alcian blue reactions of acidic glycoconjugates with phosphotungstic acid in electron microscopy

For the effective visualization of acidic glycoconjugates in electron microscopy, a post-embedding staining method has been devised for intensifying their alcian blue (AB) reactions by means of phosphotungstic acid (PTA). Tissue samples were prepared by glutaraldehyde-paraformaldehyde fixation of pieces of the trachea, aorta, and colon from adult rats. LR-White resin-embedded ultrathin sections were stained first with AB (pH = 1.0 or 2.5) and then reacted for PTA. In the tissues examined, the AB reaction of acidic glycoconjugates involved was effectively intensified by subsequent PTA staining in nearly all of the ultrastructures known to contain such carbohydrates. The majority of these ultrastructures failed to show any pronounced densities, if stained singly with PTA under the identical staining conditions. In all the ultrastructures, a series of selective methods such as active methylation and digestion with testicular hyaluronidase or neuraminidase have substantiated the selectivity of the PTA intensified AB reactions for acidic glycoconjugates involved. The present PTA intensified AB method resulted virtually in no contaminations of the backgrounds and can be regarded as a reliable and useful technique for the effective visualization of both intra- and extracellular acidic glycoconjugates in electron microscopy.     

Cytochemistry and distribution of polysaccharides in an electroreceptor: the tuberous organ of Gnathonemus petersii (Mormyrids).

The polysaccharides were studied in an electroreceptor organ, the tuberous organ of Gnathonemus petersii (Mormyridae). Histochemical methods (P.A.S., alcian blue, toluidine blue and iron colloidal reactions) allowed us to demonstrate the existence of glycogen in the sensory cytoplasm, and P.A.S. positive polysaccharides in the sensory cavity. The polysaccharides were shown to be amylase proof; they display an acidity due to the existence of sulphated radicals. The histochemical study was completed by a cytochemical analysis: a treatment with thiocarbohydrazide (TCH) according to the Thiery's method. This method allowed us to estimate the glycogen concentration, its localization, and relationship with cellular organites within the sensory cytoplasm, as well as to differentiate the highly glycogenous type II cells of the platform from the other accessory cells (Derbin and Szabo, 1968). After a treatment for 20 hours with TCH, silver stained grains were visible on the polysaccharide filaments of the sensory chamber, between the microvilli and the vacuoles of the epidermal cells lining to the sensory cavity. Silver grains coated the outer surface of the microvilli. Such polysaccharides were not identical to the filamentous polysaccharides of the cavity. In order to determine the cytochemical localization of the polysaccharide acid groups, sections were stained with iron salts. The colloidal iron constitutes a deposit opaque to electrons and located both on the filamentous polysaccharides of the sensory cavity and in the vacuoles of the epidermal cells, indicating that only these filamentous polysaccharides display acid radicals.     

Histochemical study on the bile duct system of normal Mongolian gerbils

The bile duct system of normal Mongolian gerbils was examined histochemically. The luminal surface membrane and apical cytoplasm of the biliary and gallbladder epithelial cells were stained with periodic acid-Schiff (PAS), alcian blue, pH 2.5 (AB) and high iron diamine (HID)-AB, and many epithelial cells of the common bile duct and gallbladder had weakly PAS-positive granular material in their supranuclear cytoplasm. Lectin-histochemically, these cells had binding sites to Concanavalia ensiformis (ConA), Dolichos biflorus (DBA), Glycine max (SBA), Ulex europeas-I (UEA-I), and Triticum vulgaris (WGA). On the other hand, the periductal glandular epithelial cells were not stained by any histochemical stainings. In addition to these light microscopic findings, the electron microscopic findings based on the periodic acid-silver methenamine method and avidin-biotin colloidal gold method for DBA and WGA suggested that the biliary and gallbladder epithelial cells of Mongolian gerbils secreted mucin with terminal sialic and sulfonic acid residues and that the lectin binding activity of mucin secreted from these cells was similar to that of mucin secreted from the periductal glandular epithelial cells of mice and rats.     

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 studies on the hyaline capsule of Holopedium gibberum Zaddach (Crustacea,Cladocera)

Summary The jelly capsule of the water flea Holopedium gibberum, was subjected to histochemical procedures for the visualization of acid mucopolysaccharides, amino acids, and proteins. The affinity of the capsule for azure A, alcian blue, colloidal iron, aldehyde fuchsin, and iron diamine reagents, at low pH, indicates the presence of a sulfated mucopolysaccharide. The presence of carboxyl groups, in addition, is indicated by alcian blue affinity in the combined aldehyde fuchsin-alcian blue and high-iron diamine-alcian blue procedures, as well as by the restoration of weak, but definite, basophilia after methylation-saponification pretreatment. The capsule remained alcianophilic in solutions of MgCl₂ as high as 0.4 molar. Cetylpyridinium blockade was removed by KCl solutions of 0.5–1.0 molar. The periodic acid-Schiff reaction was nil to very weak, in spite of extended oxidizing periods. None of the methods used for the visualization of amino acids or proteins gave unequivocally positive results. A possible origin of the capsular material is proposed.     

Electron microscopic observations of the surface of L-cells in culture

Summary Techniques are described for the preparation of preshadowed replicas of both the upper and lower surfaces of L-cells in culture, and of cross sections of L-cells growing on a cellophane substrate. These revealed long slender microvilli, 800 to 1,100 A in diameter, projecting from both upper and lower surfaces of the cells. These microvilli were frequently observed to contact other cells and substrate, and to leave material behind on the substrate. The plasma membrane of the lower surface was separated from the substrate by an electron-lucent gap 200 to 300 A wide. The surface coat of the L-cell was visualized by staining with colloidal iron and ruthenium. Staining with colloidal iron was most intense on the surface of the microvilli. The gap between cell and substrate was intensely stained with ruthenium red. Enzymatic digestion of living cells revealed that both trypsin and neuraminidase reduced the staining of the cell coat by colloidal iron, whereas only trypsin altered its staining with ruthenium red. After trypsin treatment, fragments of an amorphous material with the staining characteristics of the cell coat were observed between the denuded cells. Treatment with ribonuclease, chymotrypsin or hyaluronidase did not affect the staining of the cell coat.     

Spectrophotometric and cytochemical studies on azures A, B and C.

The cytochemical use of azures A, B and C, propared with either N HCL and potassium metabisulphite or with sodium hydrosulphite in tissue sections were investigated. Both in situ absorption curves of nuclei stained with each of these dye-SO2 reagents as well as in vitro absorption data of acqueous solutions of the dyes are also presented. It has been pointed out that the mechanism of staining with azure A-SO2 and azure C Eosinate-SO2 is the same as that of the conventional Feulgen reaction with Schiff reagent but that of staining with azure B-SO2 is by the modified Feulgen reaction because this dye does not contain any primary amino group.     

Histochemical evaluation of glycosaminoglycan deposition in the skin

Histologic demonstration of glycosaminoglycan (GAG) deposition in the skin has been based on the use of either colloidal iron or alcian blue. To define the best technique for the determination of skin GAG content we undertook a prospective study comparing the two stains and evaluating the use of cetylpyridinium chloride (CPC) to enhance fixation. Slides were prepared from skin biopsies obtained from five patients with cutaneous mucinoses. The preparations were coded and examined by three observers. Colloidal iron staining gave a higher intensity for GAG deposits in papillary and reticular dermis. Digestion by specific enzymes identified similar GAGs with either colloidal iron, or alcian blue; however, colloidal iron made GAGs more obvious, partly due to the contrast afforded by the yellow background stain. The addition of CPC to the fixative appreciably enhanced GAG fixation without interfering with the action of enzymes. Experimentally, we confirmed this effect of CPC by determining a pronounced decrease in GAG leakage into the fixative from CPC treated human umbilical cord. We conclude that the combination of CPC fixation and colloidal iron staining gives the best definition of skin GAGs in clinical specimens.     

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.     

Localization and characterization of carbohydrates in adrenal medullary cells

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

The histochemistry of urea-unmasked glycosaminoglycans in the skin of the eel, Anguilla japonica

In the connective tissues of the dermis and subcutis of the eel skin, the histochemistry of urea-unmasked glycosaminoglycans has been studied by means of combined staining and enzyme digestion procedures. The staining procedures employed were alcian blue (AB) pH 1.0, AB pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), AB pH 2.5-PAS, high iron diamine (HID) and low iron diamine (LID) methods, whereas the enzymes used were Streptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained have shown that a substantial amount of dermatan sulfate and a relatively small amount of hyaluronic acid, chondroitin, chondroitin sulfate A and/or C were the glycosaminoglycans involved in the connective tissues of the eel skin and that the tissues were devoid of keratan sulfate.     

Ultracytochemical Modifications of Surface Carbohydrates in Fertilized Eggs of the Common Carp

The surface of the plasma membrane of unfertilized and fertilized carp eggs was examined by four cytochemical techniques, colloidal thorium, colloidal iron, ruthenium red and phosphotungstic acid stainings, to determine the carbohydrate moieties. The surface of the plasma membrane of unfertilized eggs stained only with colloidal iron, which was heterogeneously deposited: no deposits were seen on the plasma membrane near overlying cortical alveoli. In fertilized eggs, the membrane was stained by all four methods. These ultracytochemical modifications of the surface of the plasma membrane may be caused by participation of the limiting membranes of secretory organelles, probably by turnover of the inner surface of the limiting membranes. Neuraminidase treatment of fertilized eggs eliminated the deposits of colloidal iron on the surface of the plasma membrane and caused an increase in stainability with ruthenium red. Treatment with neuraminidase or trypsin prevented the staining with phosphotungstic acid.     

CDX2在胃癌及癌前病变组织中的表达及其临床意义

目的研究CDX2在胃癌及癌前病变组织中的表达及其与临床病理因素之间的关系。方法采用免疫组织化学S-P法检测CDX2在胃癌及癌前病变组织中的表达,采用阿新兰/过碘酸雪夫氏染色(AB/PAS)和高铁二铵/阿新兰(HID/AB)染色对胃黏膜肠化生进行分型。结果 (1)CDX2在正常胃黏膜组织、慢性浅表性胃炎中不表达,CDX2在肠化、异型增生及胃癌中的阳性率(81.7%、50%、48.2%)均明显高于正常胃黏膜组织(P0.05),在肠化组织中的表达明显高于异型增生和胃癌(P0.05),在异型增生和胃癌中的阳性率差异无显著性(P0.05)。CDX2的表达与肠化的分型和异型增生的分级之间均无关系。(2)CDX2在肠型胃癌中的阳性率(67.4%)明显高于弥漫型和混合型胃癌(25.7%、42.9%);CDX2的表达与胃癌分化程度呈正相关,而与淋巴结转移和临床分期呈负相关,差异均有统计学意义。CDX2阳性表达组的五年生存率明显高于阴性表达组(P=0.038);多种变量对胃癌患者术后生存时间影响的分析显示,CDX2的表达和淋巴结转移是影响胃癌预后的独立因素(P0.05)。结论 CDX2可能在胃粘膜肠化生的发生及胃癌的发生发展中起重要作用,可作为胃癌诊断、判断胃癌的恶性程度及患者预后的有用指标。     

CYTOCHEMICAL STUDIES CONCERNING THE OCCURRENCE AND DISTRIBUTION OF RNA IN PLASTIDS OF ZEA MAYS

The occurrence of RNA in plastids from etiolated and green maize leaves was demonstrated cytochemically, with both the light and the electron microscope. Etiolated leaves were allowed to incorporate tritiated cytidine for several hours and were subsequently fixed in formalin. Radioautographs of leaf sections 2 µ thick showed silver grains over the regions of the cytoplasm containing plastids. Plastids in these sections appeared intensely basophilic when stained with azure B. Both the basophilia and radioactivity were removable with ribonuclease, clearly demonstrating the occurrence of RNA in these organelles. Examination under the electron microscope of similar plastids which had been fixed in formalin revealed a particulate component in the plastid measuring approximately 170 A in diameter. This particulate component was completely removable with ribonuclease. Thus,it was concluded that RNA occurs in a particulate form in plastids from etiolated leaves. Mature plastids, when stained with azure B, did not appear basophilic under the light microscope. Nevertheless, when formalin-fixed tissues were examined with the electron microscope, the mature plastids were seen to contain particles in the stroma, identical in appearance with those visible in the plastids in etiolated leaves. Osmium tetroxide-fixed tissues were also examined with the electron microscope. Particles similar to those seen in plastids fixed with formalin were observed, although the results obtained with this fixative were variable. It is concluded that plastids from etiolated and green maize leaves contain RNA in a particulate form which resembles ribosomes.