Histochemical staining of exogenous chromium and iron with the catalytic oxidation of phenylamines

Summary A highly sensitive and specific method for staining exogenous chromium and iron in tissues is described. This method is superior to conventional complex-forming methods with regard to its sensitivity and specificity for these metals. The staining reaction is based on the metalcatalysed oxidation of phenylamines. Tissue sections were incubated in a medium containing hydrogen peroxide and phenylamines, p-phenylenediamine or phenylhydrazine. Results obtained from test-tube experiments concerning the catalytic activities of metals indicated that the staining reactions depends on the activities of metals in tissues.     

Phenylfluorone, a new staining agent with higher selectivity for tin.

In order to search for a new staining agent with higher selectivity for particular metals, the ability of fluorone derivatives to histochemically stain metals present in rat tissues was examined. Among a variety of metals tested, phenylfluorone showed intense staining only for tin. The phenylfluorone method was superior to the conventional gallein method with regard to selectivity when staining for tin among various metals in histochemical practice.     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

A New Technique for Localization of Cellulase in situ Using Silver Nitrate

A new staining technique has been developed for the histochemical localization of cellulase in plant tissues by light microscopy. The products of cellulolysis are reducing sugars which can reduce the salts of heavy metals under appropriate conditions. The present technique relies on the deposition of black silver oxide due to reduction of alkaline silver nitrate to detect cellulase in tissues.     

The Effect of Preliminary Treatment (Fixing Fluids) on Staining Properties of the Tissues

As was reported in a previous paper,¹ staining properties depend on the chemical composition of the tissues and on the strength of the dyes themselves. Applying mixtures of basic and acid dye on tissues (methylene blue, eosin Y) at different pH-values, it is possible to find differences in the isoelectric points of the nuclei and cytoplasm of different tissues. For example, the nucleus of polymorphonuclear cells of the blood consists of the most acid protein, with an isoelectric point around pH 2.5, while the nucleus of lymphatic tissues has an isoelectric point of about pH 4.0, and that of connective tissue about pH 3.4.

With a knowledge of the above, a constant method of staining at various pH-values was used to study the effect of different fixing fluids on the staining properties of the tissues. In this way it was found that many fixing fluids gave very stable compounds with tissue proteins, and that they almost permanently change the chemical composition (i.e. the staining properties of the tissues). In some instances, these changes can be easily explained from the regular chemical standpoint. For example, formalin forms inert compounds with amino groups of the amino acids of proteins and in this way it makes the tissue proteins more acid, i.e. it moves the isoelectric point of the proteins toward a lower pH-value. The same is true in the case of the polivalent acids. The bivalent heavy metals such as mercury, on the contrary, it is assumed, combine with carboxyi groups of amino acids and in this way move the isoelectric point of the proteins toward a higher pH.     

鲫鱼不同组织过氧化氢酶同工酶活性的比较

对鲫鱼的心、鳃、肉、卵四种组织进行聚丙烯酰胺凝胶电泳并用KMnO4 法染色 (定性 ) ,同时与FeCl3-K3Fe(CN) 6 系统法进行对照 ,发现两种染色法得到的酶带具有一致性。然后用钼酸铵比色法测定四种组织中过氧化氢酶的活性 (定量 ) ,发现四种组织中过氧化氢酶的活性大小依次为 :心 >卵 >鳃 >肉。     

Acid and alkaline phosphatase activities in the clam Scrobicularia plana: kinetic characteristics and effects of heavy metals

The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues (digestive gland, gills, foot, siphon and mantle) and the 'in vitro' effect of heavy metals on both types of enzymatic activity have been analysed. The optimal pH ranged between 4.0 and 5.5 for acid phosphatase activity and 8.5 and 9.5 for alkaline phosphatase activity. The apparent optimum temperature was in the 30-60 degrees range for acid phosphatase activity and in the 30-40 degrees C range for alkaline phosphatase activity. The effect of substrate concentration on enzymatic activities in the tissues showed a good fit to the Michaelis-Menten model. For both types of enzymatic activity, the highest values were found in the digestive gland. The effect of heavy metals was dependent on the tissue analysed. Mercury showed the highest inhibition in the organs/tissues and the parameters Km and Vmax were modified when the inhibitor concentration increased, thus indicating a mixed type of inhibition.     

Highly sensitive chelating agents for histochemical staining of rare earth metals

In tissues, bromopyridylazo-diethylaminophenol has been found to be capable of staining very small amounts of rare earth metals, particularly praseodymium, terbium, dysprosium, holmium, ytterbium, and lutetium. Differentiation of a target metal from interfering metals was achieved using masking agents, polyphosphates and aminopolycarboxylic acids. Accepted: 1 June 1999     

Lack of correlation between trace metal staining and trace metal content of the rat hippocampus following colchicine microinjection

Summary Following the intrahippocampal injection of colchicine, the trace metal staining with Timm's method is shown to change in the hippocampus. The histochemical examinations were supplemented with atomic absorption spectrophotometric measurement of the trace metals (Zn, Fe, Cu). It was found that intrahippocampal colchicine treatment induces the temporary disappearance of the trace metal staining of the pyramidal cells of the regio superior, while there is a considerable reduction in the staining in the granular cells of the area dentata and in their mossy fibre terminals. Simultaneously, in contrast with the histochemical results, quantitative studies on the trace metal levels showed that colchicine does not lead to evacuation of the trace metals from the hippocampal formation. The combined atomic absorption and trace metal staining investigations prove that there is no correlation between the trace metal staining and the quantitative amounts of the trace metals.Supported by the Scientific Research Council, Ministry of Health, Hungary (4-12-0303-01-0/K)     

Analysis and Human Health Risk Assessment of Arsenic,Cadmium, and Mercury in Manta Birostris (Manta Ray) Caught Along the Ghanaian Coastline

The Ghanaian marine water of the Atlantic Ocean could be said to be polluted with As and Hg. However, Cd levels do not appear to pose an environmental concern based on levels reported in tissue samples collected. Tissue samples from six Manta birostris (manta ray) used as a bioindicator were tested for these heavy metals because many people in Ghana eat this fish (aka “kako”). The pattern of bioaccumulation of these heavy metals found in the tissues of manta rays, which feed on phytoplankton, suggests that pollution has traveled deeper into the sea, which is an environmental concern and one that may require some urgent attention. Concentrations of As, Cd, and Hg were determined in kidney, liver, and muscle samples from six Manta birostris using the neutron activation analysis method. The results showed elevated levels of the metals in the manta rays. If these values are interpreted to represent daily human exposure estimates of possible human health cancer and non-cancer risks may be derived to conclude some potential risk for daily consumers of manta ray tissues. From the assessment, most of the hazard indexes for children were less than 1 except for arsenic, which was greater than 1. This means that there is for arsenic some hazard risk for children consumers of the manta ray. This is the first study to show the accumulation of heavy metals in manta ray; although the sources are not very clear, and may be due to the numerous mining activities, it needs to be investigated further. The presence of the heavy metals is very significant because the meat (muscle) is consumed in Ghana and hence there is possible accumulation in humans.     

Histochemical demonstration of aluminum and iron deposition in pulmonary bony tissues in three cases of diffuse pulmonary ossification

Diffuse pulmonary ossification is a rare condition. We examined three cases of it in Japan, and attempted histochemically to stain for deposition of aluminum and iron in bony tissues. The patients were all female, and in their mid-twenties, mid- eighties, and later teen years. One of the patients had been exposed to heavy metals in her work involving heavy-metal analyses for 18 months. Aluminum staining and Berlin blue staining for iron were performed with dewaxed, undecalcified sections of pulmonary tissues from these three cases. Interestingly, all pulmonary bony tissues from the three cases examined exhibited linear regions of both aluminum and iron deposition in the calcifying fronts or the cement lines of bones. The patient exposed to heavy metals exhibited the most severe aluminum and iron deposition, and also exhibited positive reaction for both aluminum and iron in elastic fibers of blood vessels. Foreign body granulomas with multinucleated giant cells exhibiting elastophagia were also found in this case. This phenomenon, "endogenous pneumoconiosis", appeared to have been the cause of pulmonary hemorrhage in this case, resulting in focal heavy hemosiderosis. It is of great interest that identical patterns of aluminum and iron deposition in hemodialysis patients were found in these three cases, This is the first report on histochemical demonstration of aluminum and iron deposition in diffuse pulmonary ossification, and detailed analysis of additional cases is needed.     

Metal accumulation and apoptosis in the alimentary canal of <Emphasis Type="Italic">Lumbricus terrestris</Emphasis> as a metal biomarker

The chloragogenous tissue and the intestinal epithelium of adult earthworms, Lumbricus terrestris, sampled from sites with and without volcanic activity in the Azores were submitted to hematoxylin/eosin staining, autometallography and TUNEL-test in order to quantify the radial thickness of both tissues, their relative abundance of metals and apoptosis levels. Metals were visualized, through light microscopy, as black silver deposits (BSD) mostly in the chloragogenous tissue. The lowest radial thickness values of both tissues were found in the active volcanic sites, as well as the highest BSD and apoptosis levels. The BSD extent in the chloragogenous tissue, semi-quantified by stereology, exhibited a positive correlation with the apoptosis levels and a negative one with the radial thickness of both tissues. Thus, the variation of the radial thickness of both tissues, but especially of the chloragogenous tissue, which could reflect different cellular turnover rates caused by exposure to metals, is suggested as a biomarker of effect for metal exposure in terrestrial worms inhabiting volcanic environments.     

The influence of embedding media and fixation on the postembedment ultrastructural demonstration of complex carbohydrates. III. High iron diamine staining for sulfated glycoconjugates

The high iron diamine (HID) method for detection of sulfated complex carbohydrate has been applied directly on thin sections of variably fixed tissues embedded in epoxy and nonepoxy resins. Results with postembedment HID staining in mouse intestinal epithelium are compared to those previously obtained using preembedment methods. Sections from epoxy-embedded tissues have been found to exhibit the weakest staining intensity. Intense, specific staining was obtained in tissues not postfixed with osmium tetroxide and embedded in polystyrene, polyester resins, styrene-methacrylate, and especially the styrene-Vestopal W embedding mixture. Postosmication of tissues abolished HID staining in epoxy resins and the styrene-Spurr's resin embedding mixture, but only reduced the staining intensity in tissues embedded in nonepoxy resins.     

Immunofluorescent staining of leptospires in pepsin treated histologic sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Immunofluorescent Staining of Leptospires in Pepsin Treated Histologic Sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Histochemical demonstration of heavy metal tolerance in ectomycorrhizal fungi

Summary By the use of a specific histochemical staining method evidence was obtained that tolerance to heavy metals in ectomycorrizal fungi is based on the presence of metallothionein-like proteins. The implication that tolerance in these fungi should be induced by sublethal concentrations of heavy metals has been confirmed by us. Induction of metallothionein in ectomycorrizal fungi could possibly be helpful in protection of their host plants in areas polluted by heavy metals. In comparison with biochemical methods the histochemical method is able to locate the metal tolerance and has the added advantage that it may also be applied to mycorrhizas (root and fungus).     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

SR-G-AB显示胶原、网状纤维和粘液的复合染色法

在组织细胞的染色中,为了证明双重纤维和粘液物质,通过分别选用和组合的天狼星红(Sirius)苦味酸、Comori和阿尔辛蓝(Alcian blue)醋酸染色试剂,已能够显示鼠肺组织中胶原纤维呈红色,网状纤维呈黑色,粘液物质呈绿蓝色,背景呈黄色。对比清晰,色彩鲜艳,是较为理想的复合染色方法。     

Biochemical and RAPD Analysis of Hibiscus rosa sinensis Induced by Heavy Metals

The present study was designed to assess the effects of three different metals (cadmium, lead, and zinc) at the same concentrations on Hibiscus rosa sinensis during metal uptake. The effects of different metals at the same concentrations were assessed on biomass, root-shoot length, and biochemical parameters like chlorophyll and antioxidant enzymes like SOD and CAT to establish the tolerance potential and toxic effects on plants in different metals. The accumulation of metals by plants was found to be in the following order: Zn > mixed metals > Cd > Pb, where Zn was accumulated approximately 79.6% in plant tissues. Plants removed from Cd showed more enzyme activities than the other two metals. DNA stability was investigated by a Random Amplified Polymorphic DNA (RAPD) technique, which demonstrated that the samples in Cd and mixed metal showed similar trends, whereas samples in Zn and Pb showed similar band intensity to the control. Results suggested that Cd and/or heavy metal stress influences antioxidant status and also induces DNA changes during remediation. Therefore, these studies could be a useful biomarker assay for better treatment for metals’ remediation from soil by means of phytoremediation.