Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)     

Lead acetate as a vital marker for the analysis of bone growth

The use of lead acetate as a vital stain adds another method for the study of periodic appositional patterns of hard tissues. The distinct advantage of this technique is that these tissues can be decalcified and examined histologically without loss of the marker. The following method was used in preparation of the sections shown in the text. The rats received an intravenous injection of 4 mg lead acetate/kg body weight. After sacrifice, the tissues were decalcified in 1% HCL through which H₂S was constantly bubbled. The action of the H₂S gas is to convert the lead at sites of calcification to insoluble lead sulfide. Upon completion of decalcification the sections were embedded in 30% gelatin, and frozen sections at 15–20 μ were cut. The sections were then placed in a 0.1% solution of gold chloride for ten minutes. Next, they were placed in a 5% solution of sodium bisulphate for ten minutes. Subsequently they were washed in distilled water for 30 minutes and finally fixed in a 5% solution of sodium thiosulphate. No additional staining was used. The sections were then mounted with glycerine jelly. The resulting lead lines are sharp and therefore conducive to quantitative measurements. Because of the relatively thin sections cut, histologic details can be observed.     

The Staining of Radulae

A review of the various methods of staining and mounting radulae is given. Normally the radula should be extracted with 0.5 to 1% sodium hydroxide solution, and the associated tissues removed before staining. Two staining methods are recommended for facilitating the interpretation of radulae. Newly formed teeth and the bases of older ones are well stained by saturated aqueous chlorazol black E (up to 10 minutes). A more uniformly stained specimen) in which the cusps of all but the young teeth are alone stained, may be obtained by using the “oxidation-dahlia technic”. The radula is oxidized in N/10 potassium permanganate solution until black and subsequently decolorized in saturated aqueous oxalic acid. It is then stained in 0.1% aqueous dahlia (Hofmann's violet), the staining time varying from 10 to 30 minutes, according to the material. It is then dehydrated and passed through xylene and clove oil into Canada balsam. Other mountants may be employed but glycerin jelly is only recommended for the rapid examination of unstained radulae. Several other staining methods are mentioned, and general precautions to be observed while mounting are discussed.     

Tonic activity of submucosal neurons influences basal ion transport

The influence of tonically active submucosal neurons on basal ion transport was studied using sheets of guinea pig ileum set up in flux chambers. Tetrodotoxin evoked an immediate and sustained decrease in short-circuit current that was sustained for 60 minutes compared with control tissues in which basal currents gradually decreased over time. Time-dependent changes in basal short-circuit currents in tissues treated with atropine were not significantly different from control tissues. The decrease in short-circuit current after tetrodotoxin resulted from a greater increase in net chloride absorption than sodium absorption. Changes in net sodium and chloride transport were due to an increase in the mucosal-to-serosal fluxes of these ions. The results suggest that tonic activity of submucosal neurons limits the absorptive capacity of the guinea pig ileum.     

A method for the use of Zenker-formol fixation and the periodic acid Schiff staining technique in light microscopic radioautography

Summary Although providing superior histological preservation, Zenker-formol fixation has not been utilized in radioautographic experiments, since some component of the fixative (presumably mercury) desensitizes the emulsion and thus prevents the appearance of a radioautographic reaction. Attempts to identify and eliminate the effects of this desensitizing agent led to experiments with Zenker-formol fixed tissues from rats injected with ³H-leucine or ³H-thymidine. Radioautographs of such tissues contained a brown-black precipitate and occasionally a heavy background fog, but no normal radioautographic reaction. Moreover, when the radioautographs were exposed to light, the emulsion was transparent over the tissues, indicating that a tissue-bound component had completely desensitized the emulsion. The two components of Zenker's fluid-mercuric chloride and potassium dichromate-plus the mercurous chloride which forms as a precipitate in the course of fixation, were then tested for their individual effects on the radioautographic emulsion. It was found that mercuric chloride was primarily responsible for the desensitization of the emulsion. Merourous chloride, which formed the dark precipitate in radioautographs, produced the background fog. While potassium dichromate caused some desensitization of the emulsion, in normal histological processing where tissues had a lengthy post-fixation wash, this substance was for the most part washed out.A number of attempts were made to remove the mercury by treating the Zenker-fixed tissues with solutions of iodine, sodium thiosulfate and cysteine. It was found that the most intense radioautographic reaction was obtained with the following procedure: prior to embedding, tissue blocks were treated with 0.5% iodine solution in 70% alcohol for 17 hours. Optionally, sections of this material could be given a second treatment with 0.5% alcoholic iodine for 10 minutes. Although simple iodination eliminated the mercurous chloride precipitate and the background fog, there was still no radioautographic reaction. However, subsequent treatment of sections of this iodinated material in 5% aqueous sodium thiosulfate for 5 minutes yielded an adequate radioautographic reaction. Therefore, the simultaneous removal of both mercurous chloride precipitate and tissue bound mercury required iodination followed by sodium thiosulfate. Using this procedure, a method was described for preparing tissues for radioautography using Zenker-formol fixation and the periodic acid-Schiff staining technique.This work was supported by a grant from the Medical Research Council of Canada to Dr. C. P. Leblond.     

Histochemical Use of Sodium Bismuthate

Histochemical 1,2-glycoI cleavage, similar to that obtained with periodic acid and lead tetraacetate, may be obtained with sodium bismuthate. Routinely prepared slide sections, from tissues fixed in 10% formalin, are run down through xylene and graded alcohols to water and then oxidized for three minutes in a 1% sodium bismuthate 20% aqueous phosphoric acid solution. The oxidizing solution must be freshly prepared and used immediately. Following oxidation, sections are rinsed 15 sec. in IN HC1 to remove bismuth pentoxide precipitate, a by-product of the reaction. The sections are then washed in distilled water and placed in leuco-fushsin for 10 min., or in a saturated 30%) alcoholic solution of p-nitrophenylhydrazine for 5 min. or 2,4-dinitrophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 min. if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofushsin or yellow by the nitrophenylhydrazines.     

An in situ technique to measure bacterial chemotaxis in natural aquatic environments

A simple and reliable technique to study bacterial chemotaxis in natural aquatic environments is reported. This technique uses the test chemicals in known volumes of semi-solid agar media placed in double layered, highly porous, polyester tubes. Following in situ incubation, bacteria attracted by the test chemicals are enumerated with fluorescence microscopy following acridine orange staining. Studies in an eutrophic reservoir showed that significant numbers of bacteria were attracted to D-glucose and glycine; no significant effects were observed with L-serine, sodium succinate, or sodium chloride.     

Histochemical Use of Sodium Bismuthate

Histochemical 1,2-glycoI cleavage, similar to that obtained with periodic acid and lead tetraacetate, may be obtained with sodium bismuthate. Routinely prepared slide sections, from tissues fixed in 10% formalin, are run down through xylene and graded alcohols to water and then oxidized for three minutes in a 1% sodium bismuthate 20% aqueous phosphoric acid solution. The oxidizing solution must be freshly prepared and used immediately. Following oxidation, sections are rinsed 15 sec. in IN HC1 to remove bismuth pentoxide precipitate, a by-product of the reaction. The sections are then washed in distilled water and placed in leuco-fushsin for 10 min., or in a saturated 30%) alcoholic solution of p-nitrophenylhydrazine for 5 min. or 2,4-dinitrophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 min. if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofushsin or yellow by the nitrophenylhydrazines.     

Improved nuclear staining with mordant blue 3 as a hematoxylin substitute.

For progressive staining 1 g mordant blue 3, 0.5 g iron a alum and 10 ml hydrochloric acid are combined to make 1 liter with distlled water. Paraffin sections are stained 5 minutes blued in 0.5% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely results very resemble a good hematoxylin and eosin.     

Improved Nuclear Staining with Mordant Blue 3 as A Hematoxylin Substitute

For progressive staining 1 g mordant blue 3, 0.5 g iron alum and 10 ml hydrochloric acid are combined to make 1 liter with distilled water. Paraffin sections are stained 5 minutes, blued in 03% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely resemble a good hematoxylin and eosin.     

Histochemical Use Of Lead Tetra-Acetate I. Cleavage Of 1-2 Glycols

Lead tetra-acetate acts specifically to split the carbon-carbon single bond of the 1,2-glycol linkage to produce aldehyde radicals which may then be demonstrated by means of leucofuchsin, 2,4-dinitrophenlyhydrazine, or p-nitrophenylhydrazine. Routinely prepared slide sections from tissues fixed in 10% formalin are run down to 95% alcohol, rinsed in glacial acetic acid and then treated for 2 minutes in a saturated solution of lead tetra-acetate in glacial acetic acid with 5 g. of potassium acetate added for each 100 ml. of reagent. The sections are then washed in distilled water and placed in leucofuchsin for 10 minutes, or in a saturated 30% alcoholic solution of p-nitrophenylhydrazine for 5 minutes or 2,4-dini-trophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 minutes if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofuchsin or yellow by the nitrophenylhydrazines.     

Chelating Agents as Histological and Histochemical Decalcifiers

The alterations caused by chelating agents (disodium ethylenediaminetetraacetate) used as decalcifying solutions at pH 7.0, in histological and histochemical technics have been studied comparatively. They have been controlled by the staining with hematoxylin and eosin, Gomori's aldehyde fuchsin, periodic acid-Schiff, metachromasia, and alkaline phosphatase. Their effect on the tissues was similar to that of buffered acid decalcifying solutions, such as that of Greep, Fischer and Morse (equal parts of 2% formic acid and 20% sodium citrate).

The use of 1% sodium diethylbarbiturate for 24 hr as a reactivating agent for alkaline phosphatase in the specimens treated with chelating agents is recommended.     

A compartment model to calculate time-dependent concentration profiles of topically applied chemical compounds in the anterior compartments of the rabbit eye

Hitherto, none of the existing in vitro methods has been convincingly demonstrated to be suitable as a replacement for the Draize rabbit eye irritation test. We examine the hypothesis that one reason for this is that insufficient consideration has been given to the differences in the effective concentrations at which chemicals operate in vitro and in vivo. When a chemical is applied topically to the eye, the strength of the observed irritation that it elicits depends both on its toxic potential toward cells or tissues, and its effective concentration in the tissues of the eye. Most of the existing in vitro methods are based on isolated cells or tissues, and thus may be useful in assessing the cytotoxic potentials of chemicals. However, a reliable approach to assessing the effective concentrations of chemicals within the various tissues of the eye is lacking. A simplified compartment model is presented for calculating the time-dependent, intra-ocular concentration profiles of topically applied chemicals. The model encompasses the outer surface of the eye, three distinct segments of the cornea (subdivided into the epithelium, stroma and endothelium) and the conjunctiva. Transport through the membranes of these compartments is described as passive diffusion. For the transport coefficients, rate equations are established that contain, as free parameters, the molecular size and the partition coefficient of the chemical, as well as some intrinsic membrane parameters, such as thickness, viscosity and pore density. Numerical values for the unknown membrane parameters were estimated by fitting the theoretical rate equations to measured permeability coefficients. The compartment model was applied to an independent set of 52 test chemicals compiled from the European Commission/UK Home Office validation study. The calculated passage times (required to let 95% of the chemical reach the posterior eye tissues) varied between 0.33 minutes and 50.6 minutes, and are generally much shorter than the typical duration of observed impairments in the cornea or conjunctiva. This finding suggests that short-term contacts of the eye tissues with a chemical are sufficient to elicit long-term eye irritation. An example is given, showing how the conventional approach of using in vitro endpoints as predictors of eye irritation can be improved significantly by incorporating into the prediction the calculated intra-ocular concentration of a chemical.     

An Oxidation and Adsorption Reaction for Differentiating the Endodermis and the Collenchyma

A method is presented for a study of the endodermis and collenchyma in vascular plants. The method is useful in histologically isolating these tissues, particularly where the accepted anatomical features and the usual staining reactions are not definitive. The exclusive oxidation of the leuco (reduced) form of common redox indicators to the oxidized and colored form by the endodermis may be induced by treating fresh free-hand or freezing microtome sections with various alkaline salts at pH 8.5 to 9.0. Placing sections in sodium borate or sodium selenite induces an immediate oxidation of a tincture of gum guaiac in the endodermis and after several minutes a gradual and weak oxidation in the collenchyma. Addition of glycerol to sections which show oxidation in the endodermis induces a localized adsorption of the oxidized indicator in the collenchyma. The endodermis and the collenchyma may be differentiated best by oxidation and adsorption respectively by means of sodium selenite, guaiac and glycerol in the order given. The collenchyma in roots has not been defined to date, and the reactions described introduce a new method of study for this tissue. By treating sections with boric acid a gradient in the guaiac reaction appears in the cortex.     

Trypsin-orcein banding in plant chromosomes.

A convenient and quick method using trypsin-orcein for banding plant chromosomes (O-banding) is suggested. The technique is directly applicable to meristematic tissues (e.g. root tips) and involves the treatment of root tips with 1-2% solution of trypsin either in buffer or in 0.5 N HCl for 5-10 minutes at 37 C or for 30-60 minutes near 0 C followed by staining with 1.5% acetic orcein: 1 N HCl (19:1). Dark staining bands are reproducible and species specific. These bands possibly represent specific DNA-protein-dye interaction.     

Feulgen Hydrolysis with Phosphoric Acid

Sections from rat tissues fixed in a 10% solution of formalin in 90% alcohol were treated with phosphoric acid in concentrations varying from 30 to 85% at room temperature (28°), and subsequently stained with the Schiff reagent. Intense staining of the nuclear material was obtained in 5 to 15 minutes when 40 to 75% phosphoric acid was used. The intense staining after the higher concentrations of phosphoric acid may be due to the low concentration of water present, thus minimizing diffusion. The nucleoli in the rat-liver cells were well stained, especially the peripheral portion. The nucleoli of nerve cells, however, were only faintly stained and the Nissl substance was completely negative. The accompanying plate of photomicrographs shows the nuclei stained by this method in the Graafian follicle, the liver, intestinal villi of the rat and a metastatic carcinoma in the human pituitary.     

通络益气方对博来霉素致大鼠肺纤维化的治疗作用研究

目的:研究通络益气方对博来霉素致大鼠模型肺组织病理及肺组织羟脯氨酸(HYP)的影响。方法:将36只SPF级健康6周龄Wistar雄性大鼠置于SPF级条件下,适应性试养3天后,分为3组:空白组、模型组、治疗组,每组12只。除空白组外,其余两组,在清醒状态下,放入与小动物雾化给药仪相连的30 cm×30 cm×20 cm自制玻璃箱中,雾化吸入浓度为5 g/L(50%)的博莱霉素,雾化20分钟,休息5分钟,一天连续激发2小时,连续刺激4周。空白组在同样条件下雾化吸入0.9%生理盐水,操作方法与其余两组相同。治疗组大鼠造模后第2天起给予通络益气方,通络益气方溶液按体质量给予大鼠药液1 m L/100 g灌胃,每次于雾化前1h灌胃;模型组、正常组均给予生理盐水(1 m L/100 g)灌胃。各组大鼠连续刺激4周后,用1%戊巴比妥钠(50 mg/kg)麻醉后,待其肌肉松驰、呼吸平稳后,仰面固定于专用板上,用动物专用CT进行肺扫描。各组大鼠在末次诱导后,24 h内腹腔注射1%戊巴比妥钠麻醉后,取肺组织进行HE和Masson染色,观察各组大鼠肺组织病理HE染色、Masson染色及肺组织中HYP的含量。结果:与空白组(0.76±0.06)相比,治疗组(1.11±0.13)、模型组(1.47±0.22)HYP均升高(P<0.05);与模型组(1.47±0.22)相比,治疗组(1.11±0.13)HYP明显降低(P<0.05)。结论:通络益气方能降低对肺纤维化大鼠肺组织中胶原的沉积,起到治疗肺纤维化的作用,与通络益气方抑制HYP胶原蛋白的表达有关。     

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.     

A Batch Staining Method for Brain Slices Allowing Volume Measurements of Grey and White Matter Using an Image Analyzing Computer (Quanttmet 720)

Normal formalin-fixed gelatin-embedded cerebral hemispheres were serially sliced and the 25 to 30 slices from each hemisphere were batch stained by a modification of Mulligan's method. Following washing in water the slices were immersed in Mulligan's acid/copper sulfate/ phenol solution for 20 minutes at room temperature, treated with a xylene/Polyclens mixture for 20 seconds and immediately transferred to a 2% sodium hydroxide solution for 10 seconds. Final staining was by immersion in 2% potassium ferrocyanide which was followed by washing in tap water. The grey matter was stained a brick red color while the whiteness of the white matter was accentuated. Following staining the slices were stored between sheets of black paper in 2% aqueous formalin prior to measurement of the respective areas of grey and white matter using a Quantimet 720 image analyzing computer. The method is rapid and color stable, and reduces the risk of exposure to toxic fumes by eliminating the need for hot phenol solutions. This technique is also suitable for the macroscopic demonstration and quantitation of demyelinating conditions in the brain.