大鼠不同部位疏松结缔组织撕片的制备和观察

目的　对比大鼠皮下取材和肠系膜取材制备疏松结缔组织撕片的差异;观察同一部位取材的疏松结缔组织HE染色和醛复红-亮绿-橘黄G染色差异;分析不同部位取材的疏松结缔组织两种染色方法的结果差异。 方法　Wistar大鼠腹腔注射10 g/L苔盼蓝生理盐水溶液2.5 ml,1次/d,连续3 d,分别在皮下、肠系膜取疏松结缔组织,铺片。两个部位的铺片分别采用HE染色、醛复红-亮绿-橘黄G染色。 结果　皮下取材疏松结缔组织经HE染色可见大量成纤维细胞,肥大细胞明显,巨噬细胞可见,弹性纤维和胶原纤维可见,但不明显;皮下取材疏松结缔组织经醛复红-亮绿-橘黄G染色弹性纤维呈紫红色、胶原纤维呈橙色,细胞不易着色;肠系膜取材疏松结缔组织经HE染色,可见成纤维细胞、肥大细胞、巨噬细胞明显,弹性纤维呈蓝紫色、胶原纤维呈淡红色;肠系膜取材疏松结缔组织经醛复红-亮绿-橘黄G染色,弹性纤维被染成紫红色、胶原纤维染成鲜艳的绿色,肥大细胞被染成紫红色,核呈圆或椭圆形、棕黄色,巨噬细胞清晰可见、形态不规则,胞质中可见粗大呈蓝紫色的苔盼蓝颗粒,细胞核呈圆形、棕黄色;成纤维细胞胞质无着色,核呈棕黄色。 结论　大鼠肠系膜取材制备的疏松结缔组织撕片经醛复红-亮绿-橘黄G染色能够更好的显示各种类型细胞和纤维,各结构间对比明显。     

疏松结缔组织铺片三种染色方法的比较

<正>疏松结缔组织的标本,大多采用活体注射,取皮下组织或肠系膜铺片制成.用醛品红染弹性纤维和肥大细胞呈紫色,用伊红染胶原纤维呈红色.但是,这样往往会把背景也染上红色,使胶原纤维显示不清,为探讨一种使胶原纤维与弹性纤维、肥大细胞、巨噬细胞同在一张标本上都能明显的显示出来,又能大批制片的简便方法,我们进行了多次实验,采用三种染色方法进行比较,获得了较好的效果.1 材料和方法     

制作疏松结缔组织教学铺片的探索

关于疏松结缔组织铺片的染色技术 ,近年来不少改进 ,能同时显示三种细胞两种纤维的方法也有几种 ,但是仍有不令人满意之处 ,比如巨噬细胞吞噬颗粒太少 ,显示不清楚 ,我们推测与甲醛液固定后水洗使其溶解有关 ,也可能与台盘蓝浓度有关。所以我们经过多次实验 ,作了如下探索和改进 :1将来复红染色法中的 1 %台盘蓝注射液改为 1 .5 % ;2采用了 1 0 %、2 0 %两种不同浓度甲醛固定液固定和 95 %酒精固定进行比较。结果 2 0 %甲醛效果较佳。具体操作如下 :材料和方法 :取 2 5 0 g重大鼠 ,腹腔注射台盘蓝 ,每天 1针 ,共注射 5针 ,第五针注射后两小…     

肠系膜疏松结缔组织铺片制作改良法

观察疏松结缔组织铺片是组织学实验教学的重要内容。但标本常存在较多不足 ,比如 :两种纤维对此不够鲜明 ,各种细胞成分较难区分 ,铺片厚薄不均等。本文经过反复实验 ,建立了一种简便可行的新方法。具有特异性强、效果稳定、能够显示组织的多种成分的特点。1　材料和方法1.1　取材 :健康成年大鼠 1只 ,体重 2 0 0～ 2 5 0 g,腹腔注射新配制的 0 .5 %台盼蓝生理盐水液 ,每次 5 ml,隔日一次 ,共注射 3次。于末次注射的次日 ,断头处死 ,将肠系膜于根部连同肠管剪下 ,用大头针固定肠管于蜡盘上 ,使肠系膜展平。蒸馏水洗去血液 ,Bouin氏固定液固…     

疏松结缔组织铺片法改良

疏松结缔组织分布广泛,其中含有三种纤维和六种细胞.为使一张铺片中两种纤维和三种细胞呈现不同颜色,我们对Weigeyt方法进行改良,同时选用伊红Y和番红花O显示胶原纤维和肥大细胞,又选用健康妊娠期小白鼠、并给炎症刺激.制出的铺片满意,方法稳定可靠.1 材料和方法1.1 动物和试剂(1)选用健康妊娠期小白鼠并给刀割伤一次.(2)试剂:1注射液0.5%台盼兰生理盐水.2 固定液1:9甲醛90%乙醇混合液.3染色液 碱性复红染液:碱性复红4g间苯二碱4g蒸馏水200ml.配后加浓硫酸4ml即可应用;1%伊红水溶液(染前滴几滴冰醋酸促染);1%番红花O水溶液(染前滴几滴苯胺油促染).     

疏松结缔组织铺片染色新法

疏松结缔组织铺片的染色是组织学技术中的难点。常规方法存在着较多的不足 ,如 :两种纤维对比不够鲜明 ,各种细胞成分较难区分 ,铺片厚薄不均等。本文经过反复实验 ,能够显示组织的多种成分 ,特异性强 ,效果稳定 ,是为一种适用于教学和科研的简便可行的新方法。1　材料和方法1.1　动物的处理和取材　健康成年大鼠一只 ,体重2 0 0～ 2 50 g,腹腔注射新配制的 0 .5%台盼蓝生理盐水液 ,每次 5ml,隔日一次 ,共注射 3次。于末次注射的次日 ,断头处死 ,将肠系膜于根部连同肠管剪下 ,用大头针固定肠管于蜡盘上 ,并使肠系膜展平绷紧。用蒸馏水洗去血…     

疏松结缔组织铺片染色法的经验

疏松结缔组织在人体分布广泛 ,它的主要成分有三种纤维及多种细胞。我们对以往传统的方法进行改良 ,使一张铺片呈现蓝、紫、红三种颜色 ,能较好地观察两种纤维 ,两种细胞。我们选用健康成年兔子进行静脉注射 ,可以制取大批教学片 ,染色方法简便 ,效果令人满意。1　材料与方法动物与试剂 :动物选用健康雄性兔子一只 (2公斤重 )。试剂 :①注射液　 1%台盼蓝生理盐水溶液　②固定液　10 %福尔马林　③染液及分色液　Ｖｅｒｈｏｅｆｆ铁苏木精液 [苏木精 1克 ,无水酒精 2 0毫升 ,苏木精溶解后 ,再加入下液 :10 %三氯化铁水溶液 8毫升 ,Ｌｕｇｏ…     

家兔主要淋巴器官肥大细胞的分布特征

目的：探讨家兔主要淋巴器官肥大细胞（MC）的分布特征与组织化学特点。方法：将家兔脾脏及圆小囊用Camoy液和（或）4％中性甲醛溶液（NBF）固定,进行常规甲苯胺蓝染色或长时间甲苯胺蓝染色（LTB）,光镜观察。结果：家兔的圆小囊MC主要分布于黏膜上皮或黏膜下层结缔组织中,脾脏MC则主要分布于红髓淋巴细胞间,两者非胸腺依赖区的淋巴滤泡中均无MC,Carnoy液固定的家兔组织染色效果更佳,采用LTB染色可不同程度的增加MC的着染性。结论：MC都具有胸腺依赖性或T细胞依赖性,淋巴器官MC与T淋巴细胞位置关系密切。不同固定液和不同时间染色方法所获得的MC数量不同。     

疏松结缔组织铺片的制作方法

我们长期采用注射台盘兰(Trypanblue)显示组织细胞、Weigert 来复红显示弹力纤维(1898年)与偶氮卡红显示胶元纤维。以及用细胞质与细胞核等成份相结合的染色法作疏松结缔组织铺片的教学标本。而肥大细胞则需单独制片观察。     

显示肥大细胞的一种改良方法

显示肥大细胞的一种改良方法陈德英，郑世彬第三军医大学重庆６３００３８历来肥大细胞染色标本是组胚教学常用标本，传统制作方法是用美蓝、中性红等染结缔组织显示肥大细胞做体外活体或铺片封固观察，虽然颗粒显示清楚，却极易褪色，不易保存，且难以大量制作，不适应教...     

Staining efficacy assessment of a differential routine and special stains for pathological stromal calcifications in maxillofacial lesions

ABSTRACT

Head and neck connective tissue lesions may have diverse calcifications within the fibrous connective tissue stroma. The perplexity involved in the identification and determination of the nature or degree of calcification through routine hematoxylin and eosin (H&E) stains necessitates the usage of a specific, simple, and cost- and time-effective differential staining techniques. The aim of the present study was to develop criteria to distinguish bone formation from bone resorption using methylene blue-acid fuchsin (MB/AF) stain and the role of collagen fibers in the identification of stromal calcifications using polarizing microscopy with picrosirius red stain. Twenty cases with pathological diagnoses for various stromal calcifications in maxillofacial lesions were retrieved from the departmental archives. Decalcified formalin fixed paraffin embedded tissue sections were stained with hematoxylin and eosin, Masson’s trichrome (MT), methylene blue-acid fuchsin (MB/AF), and picrosirius red. The stained sections were assessed to identify the calcifications found in the surrounding connective tissue stroma. It was observed that most cases showed maximum staining intensity with MB/AF stain as compared to the other staining methods. Moreover, the results suggested that contrast between calcification and stromal soft tissue was best distinguished with the MB/AF stain except in the case of dystrophic calcifications. Along with this, polarizing microscopy with picrosirius red enables better characterization of stromal components. Although the H&E stain and a connective tissue stain i.e. Masson’s trichrome, are employed routinely in histopathology; the use of special stains such as MB-AF and picrosirius red facilitates the identification of calcifications from the stromal tissues.     

Detection and analysis of aldehyde fuchsin positive fibers in the connective tissue of the human oviductal mucosa II. On the biomorphosis and structural identification of the morphological substratum (author's transl)

As a result of our own investigations on the biomorphosis of the human uterine tube it is permitted to establish that the connective tissue of the lamina propria mucosae consists of 2 stainable and histochemical different types of fibers showing age-dependent changes in distribution, arrangement and localization. On the one side it is possible to detect aldehyde fuchsin positive fibrous structures (preliminary investigations and demonstration of ones were published in SCHULTKA [1980]). These fibers can be regarded as sulphur containing scleroproteins relating to the histochemical findings. The aldehyde fuchsin positive mucosal fibrous formations arising from mesenchymal tissue during the embryonal-fetal period extend into the tubal muscular system and form a subepithelial membrane-like fibrous network. In neonatal uterine tubes the fibers spread over the mucosa as a multidimensional network, and they have a intimate contact with the epithelium. Reaching the period of the fertile age the mucosal folds contain a fine branching network. Contrary to this finding, in organs of being aged women considerable thickened fibrous structures are mainly localized near the epithelium. On the other side picrofuchsin-positive connective tissue fibers, whose tinction is visibly reduced after collagenase-digestion, are localized in the tubal mucosa. Although the increase of a coarse-fibrous picrofuchsin-stainable connective tissue can be observed in the tubal folds during the 2nd half of the 4th decade of life, these morphological changes are characteristic of organs in the postmenopausis and in the senium. Finally, 2 different reacting parts of connective tissue are formed, firstly, a aldehyde fuchsin positive connective tissue localizing near the basis of the epithelium, and, secondly, a strongly picrofuchsin-positive connective tissue filling the folds, especially perivascularly. It is discussed whether, with respect to the fibrous structures selectively detected in the human mucosa, it could be the question of distinct types of collagen fibers, in which both types would represent the morphological substratum of the type III-collagen (aldehyde fuchsin positive pepsin- and papain-sensitive disulfide bonds-containing fibers) and of the type I-collagen (picrofuchsin--positive fibers), respectively. Besides, functional importance of the aldehyde fuchsin positive fibers is discussed.     

Use of Heat to Improve Connective Tissue Stains: Modifications of Masson,Movat, and Fibrin Stains

Abstract

Three connective tissue methods are presented: modifications of Masson trichrome, Movat pentachrome and a fibrin method. A modified Verhoeff hematoxylin preheated and applied in the 60°C paraffin oven was used for all methods. The Movat pentachrome modification additionally included staining with alcian blue before application of Verhoeff hematoxylin, and the fibrin method was stained with lissamine fast yellow before application of the working red stain. All sections were stained with a working dilution of Biebrich scarlet and acid fuchsin, rinsed, and differentiated with phosphotungstic acid in a 60°C paraffin oven. Demonstration of collagen in the modifications of Masson and fibrin was done with either light green or aniline blue; saffron was used in the Movat pentachrome.

All 3 techniques were improved in quality and precision with the aid of heat. Although fibrin was demonstrated in all techniques, minute quantities were better seen in the fibrin stain because the red cells were stained in a different color. These modified stains demonstrated several entities in a single slide preparation in about 20 min. (The J Histotechnol 16:349, 1993)     

Light and electron microscopic study of the eyelids,conjunctiva-associated lymphoid tissue and lacrimal gland in Bilgorajska Goose (Anser <Emphasis Type="Italic">anser</Emphasis>)

Normal structure of the accessory organs of the eye is essential for normal eye physiology. Among the most important accessory organs of the eye are the eyelids, the conjunctiva-associated lymphoid tissue (CALT) and the lacrimal gland (LG). The aim of this study was to demonstrate the histological structure of the eyelids and LG by histochemical and ultrastructural analysis. The study was performed on 13 adult female Bilgorajska geese. Eyelid samples were stained with the Alcian blue (AB pH 2.5) and periodic acid-Schiff (PAS) methods. Staining methods used for LG were AB pH 2.5, aldehyde fuchsin (AF), PAS and Hale’s dialysed iron (HDI). Within the connective tissue of the eyelids, well-developed, diffuse, CALT follicles were observed, mostly under the conjunctival epithelium. Numerous lymphocytes were present within loose connective tissue. Staining of the eyelids with the PAS method demonstrated the presence of goblet cells of a mucous nature, and AB pH 2.5 staining indicated the presence of sulfated acid mucopolysaccharides. PAS staining of LG revealed the presence of secretory cells containing weakly PAS-positive granules. All epithelial cells of the corpus glandulae and the duct systems reacted positively to AB pH 2.5. HDI staining detected the presence of carboxylated acid mucopolysaccharides. Transmission electron microscopy investigations revealed two types of secretory epithelial cells in LG. Both types of LG cells contained drop-like secretory vesicles of different sizes with low or high electron density in cytoplasm, as well as small and large lipid vacuoles, and numerous small primary lysosomes.     

Perineuronal nets of proteoglycans in the adult mouse brain, with special reference to their reactions to Gömöri's ammoniacal silver and Ehrlich's methylene blue

As our previous studies have indicated, many subsets of neurons in the vertebrate brain possess a sulfated proteoglycan surface coat which reacts to cationic iron colloid and aldehyde fuchsin. The present study demonstrated that this surface coat is supravitally stained with Ehrlich's methylene blue, and doubly with this blue and aldehyde fuchsin, a finding suggesting its being identical to Cajal's superficial reticulum (red superficial) and to Golgi's reticular coating (revetement reticulare). The perineuronal surface coat was further stained with G?m?ri's ammoniacal silver, and doubly with this silver and cationic iron colloid. These neurons with such a proteoglycan surface coat usually expressed cell surface glycoproteins which were labeled with lectin Wisteria floribunda agglutinin. Hyaluronidase digestion did not interfere with this lectin labeling of the glycoproteins, methylene blue and G?m?ri's ammoniacal silver staining of the surface coat, while it erased the cationic iron colloid and aldehyde fuchsin staining of the surface coat. These findings suggest that the perineuronal proteoglycan surface coat is associated with some additional molecules which are resistant to hyaluronidase digestion and stainable with methylene blue and G?m?ri's ammoniacal silver. The possibility is suggested that these molecules might represent "ligand proteoglycans" connecting the perineuronal proteoglycans and cell surface glycoproteins.     

Elastin VIII

Summary Nigrosin base in an acid alcohol solution and Gomori's aldehyde fuchsin gave excellent staining of the elastic fibers in the arteries and skin regardless of age. Neutral hydroalcohol solutions of alcohol soluble nigrosin stained the elastic fibers in the arteries and skin of humans above age 20. Clara's neutral hematoxylin stained the arterial elastica of children less than 10 years of age, but did not color the elastic fibers of the skin. By these staining procedures, it may be possible to obtain information about arterial elastica by a skin biopsy.     

Structure of the rat subcutaneous connective tissue in relation to its sliding mechanism

Mammalian skin can extensively slide over most parts of the body. To study the mechanism of this mobility of the skin, the structure of the subcutaneous connective tissue was examined by light microscopy. The subcutaneous connective tissue was observed to be composed of multiple layers of thin collagen sheets containing elastic fibers. These piled-up collagen sheets were loosely interconnected with each other, while the outer and inner sheets were respectively anchored to the dermis and epimysium by elastic fibers. Collagen fibers in each sheet were variable in diameter and oriented in different directions to form a thin, loose meshwork under conditions without mechanical stretching. When a weak shear force was loaded between the skin and the underlying abdominal muscles, each collagen sheet slid considerably, resulting in a stretching of the elastic fibers which anchor these sheets. When a further shear force was loaded, collagen fibers in each sheet seemed to align in a more parallel manner to the direction of the tension. With the reduction or removal of the force, the arrangement of collagen fibers in each sheet was reversed and the collagen sheets returned to their original shapes and positions, probably with the stabilizing effect of elastic fibers. Blood vessels and nerves in the subcutaneous connective tissue ran in tortuous routes in planes parallel to the unloaded skin, which seemed very adaptable for the movement of collagen sheets. These findings indicate that the subcutaneous connective tissue is extensively mobile due to the presence of multilayered collagen sheets which are maintained by elastic fibers.     

Combined staining of elastic and collagen fibers for histopathological and differential diagnosis in aorta pathologies

Histochemical techniques are important for diagnosis of connective tissue fibers involved in different aorta pathologies. The aim of this study was to develop a histochemical staining method that identifies both elastic and collagen fibers simultaneously in the same section of aortic wall. Fragments of aorta from deceased dogs were processed according to standard histological technique for paraffin embedding. Sections were stained with orcein with counterstained with either light green or single step Gomori trichrome, and also orcein stain and picro-sirius red. Orcein stain combined with picro-sirius red allowed visualization of both elastic fibers (brown) and collagen fibers (red) with light microscopy using bright field illumination. Moreover, under polarized light microscopy only collagen fibers were detected, appearing as reddish birefringent fibers, confirming that picro-sirius red-stained fibers were collagen fibers. The combination staining by orcein and picro-sirius solutions provided visualization of both collagen and elastic fibers in the same section, facilitating identification of these connective tissue fibers in the aortic wall.     

Luxol Fast Blue MBSN-Levafix Red Violet E-2BL. A combined stain for myelin sheaths and glia fibers.

During investigations of reactive dyes, Levafix Red Violet E-2BL was found suitable for staining of glia fibers. Experiments were carried out on 37% formaldehyde-fixed human autopsy material. Paraffin sections were treated with Luxol Fast Blue MBSN as usual, differentiated until glia fibers were decolorized, and counter-stained in a 0.25% solution of Levafix Red Violet E-2BL in 0.25% acetic acid. Myelin sheaths were colored blue. Gila fibers, smooth muscle cells, and nuclei were stained red violet. Axons and connective tissue remained unstained; occasionally, coarse bundles of collagen showed patchy coloration. Polarization microscopic studies proved that Levafix Red Violet E-2BL is bound to well-oriented fibrous proteins in glia fibers. The similar staining and polarization microscopic properties of glia fibers and smooth muscle support previous findings that glia fibers contain a myosin-like protein.