免疫胶体金在标记超薄切片中抗原的几点体会

金标法是利用胶体金在碱性环境中带负电的性质 ,使其与抗体相吸引 ,从而将抗体标记。我们在使用免疫胶体金标记超薄切片抗原的实验中深深地体会到 ,要提高标记阳性的成功率 ,某些因素将在实验中起到很重要的作用。1、材料和方法1 .1材料　 2 0例通过光镜、免疫荧光、电镜诊断为 Ig A肾病的肾活检组织的环氧树脂包埋块。1 .2方法　取肾活检组织 ,经 0 .0 5 %戊二醛 ,1 %多聚甲醛 (二甲砷酸钠缓冲液配制 )固定 3- 4 h,1 % Os O4后固定 2 h,梯度丙酮脱水 ,Epon81 2包埋 ,6 0℃聚合 1 2 h,制备超薄切片 ( 5 0 - 70 nm) ,粘贴于带有 formar膜的…     

胶体金技术及其发展和应用

本文主要介绍了胶体金技术的基本应用及其发展,包括应用胶体金在同一组织切片上进行免疫组化定位、银加强胶体金标记法、使用生物素化探针进行原位杂交后用胶体金技术检测(包括振动切片的包埋前法及超薄切片杂交法)以及胶体金双标记法。这些方法都是在最初的胶体金技术的基础上发展起来的,既继承了优点,又进一步进行了改良,使之广泛应用于各种理论及临床研究课题上。本文同时还简介了应用这些新技术在各个领域中的研究成果。     

微波促进胶体金标记的电镜观察

微波ＭＷ是一种频率极高的电磁波，频率为２．５ＧＨｚ，当微波照射免疫胶体金标记切片时，切片上的抗原抗体极性分子随微波周期以每秒２４亿５千万次的速度进行快速振动，这种快速振动产生的摩擦热促进了组织细胞内的生理生化反应过程，从而加速了抗原与抗体之间的结合。在电镜下，垂体腺瘤和布鲁氏杆菌的超微结构保存较好，细胞形态无改变，在胶体金标记的抗原抗体结合处，金颗粒清晰可辩，切片背景清洁，无金颗粒弥散，很易与其它反应产物和染色反应物质相区别（见图）。微波用于免疫胶体金电镜切片标记不仅效果佳，背景清晰，而且大大缩…     

丙烯醛固定，PAP与胶体金免疫电镜双标记技术在神经细胞内抗原标记的应用

本实验采用了PAP和胶体金免疫电镜双标记技术,对下丘脑室旁核神经元內进行标记,证实了室旁核内有血管加压素与生长抑素共存的神经元.为了加强抗原的固定效果,提高免疫反应的敏感性,我们采用了丙烯醛固定的方法,对比了浸泡固定和灌流固定的效果,在电镜水平证实了丙烯醛浸泡固定同灌流固定一样,均能获得良好的神经组织的超微结构和阳性反应。同时,我们分别采用了包埋前PAP与胶体金双标记和包埋前PAP结合包埋后胶体金双标记两种方法。结果表明,包埋后胶体金染色比包埋前较易发现阳性标记。     

包埋前免疫金电镜标记的使用难点及解决方法

根据免疫标记与标本之间的不同关系,免疫电镜技术可分为包埋前、包埋后及冷冻超薄切片免疫电镜技术。本文仅介绍包埋前免疫金电镜标记在使用过程中的一些难点及解决方法。     

微波、高压、真空负压和电炉四种加热法在抗原修复中的比较

常规免疫组织化学染色难以使癌基因、抑癌基因及其产物显示满意的效果。应用高温对组织抗原进行修复，可有效地暴露抗原决定簇而增强免疫组织化学染色的结果t‘］。据此，我们应用T4种不同的加热法，对胶质瘤组织抗原的修复进行了对比研究。l材料和方法1．l材料为我科收集的外科检查确诊为胶质瘤的标本，共80例，均经10％福尔马林液固定，石蜡包埋。每例做连续4M切片20张，附贴于涂有多聚一L一赖氨酸的玻片上，置60t烤箱内烘烤4h。P16，Rb，EGFR，CyclinDI及S－P试剂盆，均为美国饲edmBiotech公司产品；多聚一L一赖氨酸购自福州通新…     

免疫组织化学展望

尽管免疫组织化学技术发明于20世纪40年代,但用于甲醛固定石蜡包埋组织切片的历史仅有30余年.一系列的技术发明,包括在酶标记抗体的基础上开发各种高敏感性检测系统(如PAP,ABc等),单克隆抗体的问世以及蛋白酶消化修复部分抗原等,为免疫组织化学技术用于石蜡切片提供了有利条件.     

微波在免疫电镜胶体金技术中的应用

微波在免疫电镜胶体金技术中的应用姜秀英，赖晃文，刘旭明，邹赛英关键词胶体金标记，微波，免疫电镜中国图书分类号Ｒ４４６．８当微波照射免疫胶体金标记切片时，切片上的抗原抗体极性分子随微波周期以每秒２４亿５千万次的速度进行快速振动，这种快速振动产生的摩擦热...     

应用包埋前免疫胶体金直接法研究细胞内病毒抗原超微定位

目的：探讨细胞内病毒抗原的超微定位的方法。方法：采用包埋前免疫胶体金方法标记Ⅰ型单纯疱疹病毒（ＨＳＶ－１）抗原以研究该病毒在人胚肺细胞（ＨＥＬ）内的超微定位。结果：发现金颗粒约为５ｎｍ，大小均一，在ＨＳＶ－１囊膜、内质网膜表面及其近区域 有大量的金颗粒，而阴性对照在这些地方未峁金颗粒。结论表明包埋前免疫胶体金方法可靠，可用于病毒抗原的超微定位研究。     

抗原修复技术研究发展的新起点

7年多前，我们曾经受贵刊编辑部之邀发表过“抗原修复技术研究进展”一文［1］。在那篇文章里，尽管已经有片言只语提到可以应用抗原修复技术原则从石蜡切片中提取蛋白质和核酸。但是，全文的主题与绝大部分内容还是局限在免疫组织化学染色的问题方面。转瞬过去的7年在后基因组时代的舞台上大放异彩，高通量检测数以千计的蛋白质组学分析技术的开发以及各种形形色色的蛋白质质谱分析技术的开发，在探讨人类疾病的多种相关生物标志物及其在诊断治疗上，正在扮演着先锋作用的重要角色［2-4］。图像质谱分析是最近开发出来的一门新技术，它是建立在质谱分析法特别是通过基质辅助激光解析（ matrix-assisted laser desorption ionization ）技术或者次级离子质谱分析法（ secondary ion mass spectrometry ）能够在一张组织切片上进行蛋白质、脂质等细胞组成成分在组织结构内的质谱分析。1997年，离子质谱分析法（ IMS ）最早由Caprioli的实验室发表。此后不久即开始用于临床研究，如癌组织中HER2表达以及分布等的研究显示出颇具魅力的发展前景。但是，这些新兴的技术从开始起步就面临着和20多年前免疫组织化学在常规甲醛固定石蜡包埋（ FFPE ）组织切片染色上同样的尴尬局面：必须依赖新鲜组织冷冻切片。随着抗原修复技术在石蜡切片免疫组织化学染色的广泛推广，人们从数以万计的近代有关加热抗原修复技术的文章中，毫不怀疑地认识到，加热抗原修复技术在免疫组织化学染色等方面取得的优异成果，开启了如何把现代分子生物学技术用于FFPE组织科研宝库的途径。继免疫组织化学染色之后，加热抗原修复技术的基本原理已经成功地被用于包埋前、后免疫电镜染色，核酸原位杂交，脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记，从石蜡包埋组织中提取蛋白质或核酸等一系列的重要成绩。2008至2011年期间，意大利的Ronci等［5］、澳大利亚的Gustafsson等［6］、美国的Casadonte和Caprioli［7］相继发表了他们经过反复试验、成功地采用加热抗原修复技术方法，使得IMS能够用于常规FFPE组织切片的文章，由于从事IMS技术开发的科学家们逐渐从与日俱增的加热抗原修复在免疫组织化学染色等方面令人瞩目的成绩中，认识到FFPE人体组织是不可取代的极为宝贵的科学研究资源，任何一种现代化的分子生物学技术都不能够仅仅局限于新鲜组织冷冻切片。必须千方百计地寻求一种能够克服FFPE处理给予组织内高分子成分带来的负面影响的有效方法。于是，一切从事与常规FFPE组织标本相关联的科学研究和技术开发都顺理成章地归结到加热抗原修复技术。有力地证明这一极为简单的加热修复方法的确提供了解放石蜡组织中的蛋白质等高分子结构成分的坦途，开启了为病理学家和形态学家步入分子病理学、形态学的大门。     

Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy

To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electron-dense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-labelling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10-100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.     

Comparison of different methodologies and cryostat versus paraffin sections for chromogenic immunohistochemistry

Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.     

An enhanced immunocytochemical method for staining bone marrow trephine sections.

AIMS: The detection of cellular antigens in fixed decalcified bone marrow trephine (BMT) sections depends on the method of processing, the nature of the antigen and antibody, antigen retrieval techniques, and the sensitivity of the immunocytochemical method. This study evaluated a tyramide enhanced avidin-biotin immunostaining method on formalin fixed decalcified BMT sections to determine whether the method could detect previously undetectable antigens. METHODS: Nineteen BMT biopsies from a range of haematological disorders were evaluated with 43 antibodies to haemopoietic antigens using horseradish peroxidase and alkaline phosphatase detection methods, using the tyramide enhanced avidin-biotin immunostaining method. RESULTS: Compared with standard avidin-biotin immunostaining methods the tyramide enhanced immunostaining method showed enhanced signal intensity, gave positive labelling for antigens that require pretreatment by other methods, and previously unreactive antigens were detected. Primary antibodies could be used at up to 200 times higher dilutions. CONCLUSION: The tyramide enhanced immunostaining method, while retaining specificity, is highly sensitive and enables an increased number and range of antigens to be detected than previously possible. The method could be applied to BMT sections for the routine diagnosis and classification of haematological disorders.     

Application of methods of heat retrieval of antigens in paraffin sections of rat brain

The aim of this study was to develop an optimal protocol of processing of rat brain sections permitting to minimize the shortcomings of the method of heat retrieval of antigens and preserving its high efficiency. The research was conducted using the methods of heat antigen retrieval, immunohistochemistry and light microscopy. It was shown that prolonged incubation in blocking solution, containing normal serum of an animal--source of secondary antibodies, eliminated false-positive reaction, demonstrated after heat antigen retrieval in paraffin sections of rat brain.     

Immunogold-Silver Staining of Immune Deposits in Renal Biopsies

Abstract

A procedure that facilitates the light and electron microscopic demonstration of immune deposits in renal biopsies with immunogold staining is described. Examination of a single specimen is achieved with both light and electron microscopy when LR White acrylic resin is used. Minimal aldehyde fixation and low temperature processing retains maximum antigen availability. For light microscopy, an indirect immunogold silver system is applied to 2μm LR White sections, which reveal immunoglobulins and complement C3 in the familiar pattern associated with various glomerular diseases. Indirect immunogold is applied to ultrathin sections prepared from the same block to demonstrate labeling of the same antigens at the ultrastructural level. (The J Histotechnol 16:243, 1993)     

Cell surface and intracellular expression of two Candida albicans antigens during in vitro and in vivo growth

Intracellular and cell surface expression of two antigenic determinants of Candida albicans was studied by reacting ultrathin sections of the fungus with monoclonal antibodies and locating the sites of antibody-antigen reactions by use of colloidal gold-coupled secondary antibody and electron microscopic techniques. Comparisons were made between fungal cells allowed to develop in vitro and cells (in vivo) recovered from infected mice. Differences between in vitro antigen expression and expression in vivo were particularly noted during early germination events. Antigen expression in vivo appears to be concentrated in deeper cell wall layers and beneath the plasmalemma especially on germ tubes. Cells which develop in vivo and in vitro have antigen associated with an outer flocculent layer and on the innermost cell wall layer of mother cells and hyphae. A middle electron transparent cell wall layer is usually devoid of antigen. Comparison of antigen expression during in vivo growth in early (intraperitoneal) infection versus late disseminated (kidney) disease suggests that both of the antigens are expressed in greater quantity on the surface of germ tubes than on mother cells in kidney tissue as compared to intraperitoneally-grown cells. Results also suggest that at least one of the antigens, which is a substituted mannan, can be found on a lamello-tubular intracellular structure.     

Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.     

Hepatocyte localization of hepatitis B core and surface antigens in renal transplant recipients

Summary A prospective series of 45 liver biopsies taken from 22 renal transplant patients was investigated for the presence of hepatitis B antigen core (HBc) and surface (HBs) components by electron microscopy. At the time of each biopsy serum HBs Ag was sought by radioimmunoassay. Sections were taken for the detection of HBs Ag by immunofluorescence.In seropositive patients, intravesicular tubular structures resembling HBs Ag were found in 61% of biopsies while the intranuclear core HBc was present in 69%. No correlation could be made between the ultrastructural pattern of the viral components and the intensity of the histological liver damage. During the follow up, there was an accumulation of both HBs and HBc Ag even in a period as short as 1 year. The 9 liver specimens examined after three years of transplantation showed a marked accumulation of both antigens. Thus the expression of HB Ag at the hepatocellular level seems to correlate better with the duration of antigenaemia than with the histological pattern.Lastly, on matched semithin and ultrathin sections, the ground glass appearance of cytoplasm appeared to correlate with smooth endoplasmic reticulum distorsion, irrespective of the simultaneous presence or absence of intravesicular tubular structures. The sanded nuclei expressed a rare massive accumulation of core antigen.     

Detection by immunogold techniques of HIV antigens in Lowicryl ultrathin sections of infected cells

A post-embedding technique for immunocytochemical analysis at the ultrastructural level was used to detect and localize HIV antigens on ultrathin sections of Lowicryl K4M-embedded HIV-infected cells. With serum from an AIDS patient, specific immunogold labelling was obtained exclusively on mature viral extracellular structures. The more intense reactivity was obtained with core antigens. The present immunoelectron microscopy method provides several advantages - high sensitivity of immunodetection, good preservation of cellular morphology, easy preparation procedure - which could lead to the use of this method for HIV-infected human tissues.     

用于甲醛固定石蜡包埋组织的改良免疫荧光法***

背景：甲醛固定石蜡包埋的标本易于保存,免疫荧光法定位定量能精确支持多种标记物。 目的：建立适合于甲醛固定石蜡包埋组织的改良免疫荧光法。 方法：将取自类风湿性关节炎和无骨性关节炎患者的滑膜血管翕和髋臼增生组织的石蜡切片进行常规脱蜡复水,观察不同的柠檬酸缓冲液热修复时间、血清封闭时间、一抗浓度和荧光标记物在免疫荧光法中显色的差异。 结果与结论：柠檬酸高温修复15 min后滴加抗原修复液比单纯高温修复10~20 min,组织结构保存更为完整,且背景染色无显著区别;选择体积分数10%二抗来源正常血清室温封闭40 min作为最佳的抗原封闭条件,既可以保证良好的封闭效果,同时又不影响一抗和抗原位点结合;较高一抗浓度(10 mg/L)能保证明显的荧光信号、低背景和可接受的成本。进行石蜡切片免疫荧光实验,并用普通荧光显微镜观察,应避免采用FITC等蓝色激发光、呈现绿色荧光信号的标记染料基团。提示柠檬酸高温修复15 min后滴加抗原修复液,选择体积分数10%二抗来源正常血清室温封闭40 min,较高一抗浓度,普通荧光显微镜观察可提高免疫荧光法的检测质量。