Trichrome Staining for Detection of Amyloid

It has been proposed to use trichrome staining of histological sections for the detection of connective tissue fiber and sites for amyloid localization, as well as for increasing color contrast. After incubation in acidin–pepsin solution, sections are dewaxed and successively stained with picrofuchsin according to van Gieson, together with nuclei counterstain with hematoxylin, Congo red, and picroindigocarmine. As a result, the amyloid bound with collagen fibers was stained brick-red, collagen and reticular fibers not bound with amyloid was stained blue-green, and cytoplasm of cells not containing amyloid was stained yellow. Trichrome staining of organs affected by amyloidosis is more informative for the analysis of organs than Congo red stain.     

Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon

Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon.     

Comparison of methods for staining microvessels in bone

Detection of microvessels is critical for studying bone tissue. We developed an intravascular ink-based method coupled with Van Gieson (VG) staining and compared it with other commonly used methods for capillary visualization. The ink perfusate was formulated as 10% ink, 10% formaldehyde and 20% mannitol. The ink solution was perfused into a healthy goat and the tibia was subjected to decalcification, dehydration, paraffin embedding, de-waxing and staining to observe microvessels. Angiogenesis was assessed by vascular area image analysis and the hematoxylin and eosin (HE), Masson, and VG staining techniques were compared to determine the reliability of these methods for counting microvessels. We found that HE, Masson, and VG staining produced poor contrast between the microvessels and surrounding tissues. By contrast, ink coupled with VG staining permitted clear discrimination between the microvessels and surrounding tissues. Our results indicate that ink-VG staining could be more useful than other methods for detecting tissue microvessels.     

Comparison of methods for staining microvessels in bone

Abstract

Detection of microvessels is critical for studying bone tissue. We developed an intravascular ink-based method coupled with Van Gieson (VG) staining and compared it with other commonly used methods for capillary visualization. The ink perfusate was formulated as 10% ink, 10% formaldehyde and 20% mannitol. The ink solution was perfused into a healthy goat and the tibia was subjected to decalcification, dehydration, paraffin embedding, de-waxing and staining to observe microvessels. Angiogenesis was assessed by vascular area image analysis and the hematoxylin and eosin (HE), Masson, and VG staining techniques were compared to determine the reliability of these methods for counting microvessels. We found that HE, Masson, and VG staining produced poor contrast between the microvessels and surrounding tissues. By contrast, ink coupled with VG staining permitted clear discrimination between the microvessels and surrounding tissues. Our results indicate that ink-VG staining could be more useful than other methods for detecting tissue microvessels.     

多种特殊染色法在骨关节炎组织形态学研究中的应用比较

目的探讨多种特殊染色法在骨关节组织中的染色规律及其在骨关节炎形态学研究中的应用价值。方法 6月龄健康新西兰大白兔20只,随机分为正常组和造模组各10只,根据改良Hulth法造模,6周后膝关节取材。对标本固定、脱钙后进行石蜡包埋和切片。分别采用HE、番红-固绿、AB-PAS、甲苯胺蓝、Van Gieson染色和Mallory染色,观察骨关节组织的形态学变化,并对几种染色方法进行比较。结果 HE染色显示关节一般组织形态结构,可见模型组关节软骨和软骨下骨发生骨性关节炎病理变化;番红-固绿染色法中软骨和软骨下骨的界限（黏合线）以及潮线显示清晰,软骨基质中糖胺聚糖含量减少,纤维成分增多;AB-PAS染色显示骨关节炎软骨基质糖胺聚糖尤其是酸性糖胺聚糖含量减少;甲苯胺蓝染色显示骨关节炎软骨的酸性糖胺聚糖减少;Van Gieson染色和Mallory染色可显示骨关节组织中的胶原纤维,但组织结构界限不够清晰。结论在骨性关节炎的组织形态学研究中,通过常规HE染色,结合番红-固绿染色法和AB-PAS染色法,能较客观全面地获得关节组织形态学相关信息。     

Stain Technology: A Combined Elastic, Fibrin and Collagen Stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified VerhoefPs elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by Hssamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsia, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

A combined elastic, fibrin and collagen stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified Verhoeff's elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by lissamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsin, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

The Masson Trichrome Staining Methods in Routine Laboratory Use

A resume of Masson's trichrome staining methods is given, with detailed directions for carrying out all of his procedures. The results obtained thru their use in a routine laboratory are discussed at length, as well as the fact that they also work very well on tissues fixed in ways other than those he prescribes, and stained with chemicals and dyes other than those he uses. The fact is stressed, however, that the closer one adheres to his precepts, the better will be the results.

The stains described include bis hematozylin-phloxine-saffron, his iron-hematozylin-ponceau-anilin-blue, his variants of this stain (of which the light green stain is excellent), his metanil yellow and his modification of the familiar Van Gieson technic. All these stains are based on familiar laboratory methods, improved and rendered trichrome, so that they present no great obstacles in technic.

Of the methods cited, the writer prefers the “light green” procedure. Sections are prestained in Regaud's iron-hematoxylin, followed by a mixture of ponceau de xylidine and acid fuchsin. This is followed by mordanting in phosphomolybdic acid and the sections are finally stained in light green. The results are very precise and pleasing and afford immediate orientation as the connective tissue is green, the nuclei black or dark purple, the cytoplasm of the cells is in varying tones of red. The method may be used after fixation in almost any good medium; altho the results are not as brilliant as those obtained after one of Masson's prescribed fixations, it is believed that they are even then superior to those following the routine hematoxylin-eosin method.     

The Application of Miller's Elastic Stain to Glycol Methacrylate Tissue Sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

The application of Miller's elastic stain to glycol methacrylate tissue sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure

Abstract

A modified dichromatic iron-eriocyanine R (Fe-ECR) staining method is described. Staining obtained with this new technique generally was similar to that of hematoxylin and eosin (H & E). Cell nuclei were stained blue. Cardiac, smooth and skeletal muscle, and red blood cells, were stained different shades of red. Collagen fibers were stained different shades of orange, usually faintly. Decalcified bony tissue was stained pinkish violet. Epithelial cells were strongly stained deep shades of red, magenta and violet. Cartilage matrix, and goblet and mast cells were unstained. Although Fe-ECR staining differed too much from standard H & E staining to be a substitute for diagnostic purposes, the dichromatic method described might usefully replace van Gieson or trichrome stains, especially if muscle is of interest. A pH 0.95 staining solution was used to differentiate initially over-stained sections followed by washing in distilled water. This dichromatic technique is easier to perform and more precisely controllable than other ECR dichromatic methods. The entire procedure can be completed in less than 5 min. The technique has the advantages of greater technical simplicity and speed, a larger range of polychromasia, and a longer shelf-life than H & E. ECR also is more reliably available than hematoxylin and usually is less expensive.     

Decellularized omentum as novel biologic scaffold for reconstructive surgery and regenerative medicine

Homologous tissues, such as adipose tissue, may be an interesting source of acellular scaffolds, maintaining a complex physiological three-dimensional (3D) structure, to be recellularized with autologous cells. The aim of the present work is to evaluate the possibility of obtaining homologous acellular scaffolds from decellularization of the omentum, which is known to have a complex vascular network. Adult rat and human omenta were treated with an adapted decellularization protocol involving mechanical rupture (freeze-thaw cycles), enzymatic digestion (trypsin, lipase, deoxyribonuclease, ribonuclease) and lipid extraction (2-propanol). Histological staining confirmed the effectiveness of decellularization, resulting in cell-free scaffolds with no residual cells in the matrix. The complex 3D networks of collagen (azan-Mallory), elastic fibers (Van Gieson), reticular fibers and glycosaminoglycans (PAS) were maintained, whereas Oil Red and Sudan stains showed the loss of lipids in the decellularized tissue. The vascular structures in the tissue were still visible, with preservation of collagen and elastic wall components and loss of endothelial (anti-CD31 and -CD34 immunohistochemistry) and smooth muscle (anti-alpha smooth muscle actin) cells. Fat-rich and well vascularized omental tissue may be decellularized to obtain complex 3D scaffolds preserving tissue architecture potentially suitable for recellularization. Further analyses are necessary to verify the possibility of recolonization of the scaffold by adipose-derived stem cells in vitro and then in vivo after re implantation, as already known for homologus implants in regenerative processes.Key words: omentum, scaffold, decellularization, adipose tissue engineering, regenerative medicine, microvascularization     

实验动物组织血管铁质反应与胶原、弹性纤维和肌纤维的组合染色法

在进行实验动物组织染色反应中,为了证明血管铁质物质与胶原、弹性纤维和肌纤维的分布情况,选用丽春红S苦味酸(Ponceau S picric acid)、间苯二酚碱性复红(Resorein basic fuchsin)与Perls铁法(简称P-PA-R-BF-P法),对大鼠组织进行组合染色,能够较好显示组织血管中铁质物质与胶原、弹性纤维和肌纤维,铁质物质呈蓝色,胶原纤维呈红色,弹性纤维呈棕色,肌纤维呈黄色。这是一种相互对比清晰的组合染色方法。     

鳖源胶原蛋白提取纯化及在生物材料中的应用

近年来,胶原蛋白因其良好的生物学性能在生物材料应用中得到越来越多的关注,为了建立一种快速高效的鳖源胶原蛋白纯化方法和探究其在生物材料中的应用价值,首先用Van Gieson染色法和苦味酸-天狼星红染色法观察裙边胶原纤维组织结构,发现裙边胶原纤维含量非常高且类型主要是Ⅰ型。采用不同截留分子量的透析袋对裙边胶原蛋白粗提液进行直接透析纯化,发现截留分子量为100 k Da的透析袋在透析48 h后对裙边胶原蛋白的纯化效果最好,SDS-PAGE检验显示几乎没有杂带。对裙边胶原蛋白生物学性能包括吸水性、体外降解性进行考察,其吸水力和持水力分别高达12.06 g/g和98.21%,且在72 h后被完全降解;对裙边胶原蛋白海绵的溶血性、皮肤致敏性、肝部创伤止血性、促创伤皮肤愈合性进行了研究,并和交联胶原进行比较,发现裙边胶原蛋白和交联胶原均不会造成SD大鼠溶血和皮肤过敏,二者均具有良好的止血效果,且裙边胶原蛋白能显著缩短创伤皮肤愈合时间,具有良好的促创伤皮肤愈合性,而交联胶原效果欠佳。本研究表明裙边胶原蛋白表现出了优良的生物学性能,在生物材料领域具有很大应用价值。     

A Modification of the Fite Formaldehyde (Fite I) Method for Staining Acid-Fast Bacilli in Paraffin Sections

For maximal demonstration of acid-fast bacilli in tissue sections it is necessary to avoid sequences of reagents which, first, affect the integrity of the complex upon which acid-fastness depends, and, second, extracts it from those bacillary elements which have been made vulnerable by age or other factors. The greatest damage occurs during dewaxing of paraffin sections by xylene and alcohols; the older and more decrepit bacilli being especially affected. In the technic presented, which is a modified combination of two processes devised by Fite, sections are deparaffinized by a protective mixture of rectified turpentine and heavy liquid petrolatum (2:1) and blotted to water. Staining is with Fite's new fuchsin (magenta III) solution, overnight at room temperature. The sections are then treated with reagent grade formaldehyde, which turns the color of the bacilli deep blue-black, followed by an aqueous sulfuric acid decolorizer, the potassium permanganate-oxalic acid sequence, and a modified Van Gieson counterstain, nuclear staining with hematoxylin being omitted. For total demonstration of all stainable bacilli, restorative treatment in the turpentine-oil mixture before staining is sometimes required, most frequently with leprosy material but also with some tuberculosis lesions.     

A histochemical study of ultraviolet B irradiation and Origanum hypericifolium oil applied to the skin of mice

Abstract

Ultraviolet (UV) rays cause skin damage. Chronic exposure to UV irradiation causes decreased collagen synthesis, degenerative changes in collagen bundles, accumulation of elastotic material and increased epidermal thickness. Origanum hypericifolium, an endemic Turkish plant, belongs to Lamiaceae family. The main constituents of its oil are monoterpenes including cymene, carvacrol, thymol and γ-terpinene. The effects of undiluted O. hypericifolium oil on UVB irradiated skin of mice were investigated histochemically. Four groups of female BALB/c mice, whose dorsal hair was shaved, were allocated as follows: non-UVB irradiated (Group 1), UVB-irradiated (Group 2), O. hypericifolium oil treated (Group 3), and O. hypericifolium oil treated and UVB irradiated (Group 4). Sections of dorsal skin samples were stained with Mallory's phosphotungstic acid hematoxylin for collagen fibers and Taenzer-Unna orcein for elastic fibers. Sections also were stained with hematoxylin and eosin to measure epidermal thickness. We observed intense staining of collagen and homogeneous, scattered thin elastic fibers in Group 1; scattered and weakly stained collagen and curled, amorphous, accumulate elastic fibers in Group 2; and intense staining of collagen in Groups 3 and 4. Accumulation of elastic fibers in the dermis was unremarkable in Groups 3 and 4. In Groups 3 and 4, O. hypericifolium oil treatment thickened the epidermis. Epidermal thickness was greatest in Group 4. We suggest that O. hypericifolium oil may block UVB induced alterations of collagen and elastic fibers, and increase epidermal thickness.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Modified elastic tissue-Masson trichrome stain

A combined elastic tissue-Masson technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeff's iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

A Modified Verhoeff-Van Gieson Elastin Histochemical Stain to Enable Pulmonary Arterial Hypertension Model Characterization

Optimal histochemical staining is critical to ensure excellent quality stained sections to enable light microscopic and histomorphometric image analysis. Verhoeff-van Gieson is the most widely used histochemical stain for the visualization of vascular elastic fibers. However, it is notoriously difficult to differentiate fine elastic fibers of small vasculature to enable histomorphometric image analysis, especially in organs such as the lung. A tissue fixation procedure of 10% neutral buffered formalin with subsequent fixation in 70% ethanol further compounds the problem of small vessel staining and identification. Therefore, a modified Verhoeff’s elastin stain was developed as a reliable method to optimally highlight the internal and external elastic laminae of small arteries (50-100 µm external diameter) and intra-acinar vessels (10-50 µm external diameter) in 3 µm thick lung tissue sections from models of pulmonary arterial hypertension. This modified Verhoeff’s elastin stain demonstrated well-defined staining of fine elastic fibers of pulmonary blood vessels enabling subsequent histomorphometric image analysis of vessel wall thickness in small arteries and intra-acinar vessels. In conclusion, modification of the standard Verhoeff-van Gieson histochemical stain is needed to visualize small caliber vessels’ elastic fibers especially in tissues fixed in 10% neutral buffered formalin followed by additional fixation in 70% ethanol.Key words: Histochemical stain, histomorphology, lung, Verhoeff-van Gieson, elastin