不同抗原修复方法对肝癌组织中内源性生物素的影响及对策

目的探讨不同抗原修复方法对肝癌组织中内源性生物素的影响以及消除办法.方法采用pH6.0、pH8.0及pH9.0不同pH值的抗原修复液进行修复,对30例经甲醛固定石蜡包埋的肝细胞癌组织中内源性生物素活性进行检测,并通过不同检测方法对30例肝细胞癌中AFP(Alpha Fetoprotein)染色结果进行对比.结果加热抗原修复暴露肝细胞癌组织中内源性生物素的活性.不同pH值抗原修复液对内源性生物素的活性影响各不相同,阳性强度随着pH值增高而增强.生物素阻断剂能有效阻断内源性生物素的活性.采用pH9.0Tris-EDTA进行修复加Envision检测系统可提高肝癌组织中AFP的阳性率及阳性强度.结论加热抗原修复可使被甲醛封闭的内源性生物素的活性重新暴露,对免疫组化的染色结果造成影响,生物素阻断系统是消除内源性生物素的有效方法,采用pH9.0的抗原修复液进行修复加Envision非生物素检测系统是免疫组化染色中较为理想的检测方法.     

微波快速免疫荧光组化染色方法的研究

本文应用微波辐射方法加速免疫荧光组化染色(间接和直接法),分别定位15种不同组织抗原,并应用连续切片同时用两种不同的孵育方法即微波辐射和常规孵育方法进行比较。结果证明,经微波辐射后免疫荧光组化染色时间大大缩短,背景染色明显好于常规法,阳性率和阳性强度与常规法基本一致。     

多重PAP及多重PAP结合ABC免疫细胞化学方法的建立、敏感性分析与应用

作者在传统的 PAP 及 ABC 染色方法的基础上,通过重复使用桥抗及 PAP 的次数或重复使用生物素化桥抗及 PAP 和 ABC 的次数,使 PAP 与 ABC 方法成功地结合,增加连接于抗原部位的酶分子数,建立起两种敏感的免疫细胞化学方法。染色结果经显微分光光度图象分析,方差分析,回归分析及染色效果的直接观察表明,重复染色可增加阳性强度,扩大阳性染色范围,提高信噪比,从而提高方法的敏感性,以生物素化桥抗取代非生物素化桥抗,将 ABC 引入到多重 PAP 方法中,可使方法的敏感性进一步提高。该方法结合单克隆抗体及多克隆抗体成功地检测出用传统双PAP 及 ABC 难以或不能检测出的常规福尔马林固定石蜡包埋的肾综合征出血热尸检组织中的病毒抗原及石蜡包埋肿瘤活检组织中的桥粒抗原。     

用于免疫印迹法的生物素化标准分子量蛋白质的制备

免疫印迹技术目前已被广泛应用。确定抗原分子量通常使用标准分子量蛋白平行电泳并转移至NC膜,切下相应条带用氨基黑等染料染色,不但费时,敏感性低,而且在染色和脱色时易引起NC膜的皱缩。1986年Della-Penna等报告用生物素化蛋白作为蛋白质印迹(Western blots)的分子量标准品。目前国外已有商品出售,但价格较贵。本文利用国产低分子量标准蛋白质和自制N-羟基琥珀酰亚胺生物素酯制备生物素化蛋白取得满意结果。     

用亲和素-生物素复合物法研究流行性乙型脑炎病毒感染小鼠中枢神经系统后的抗原定位

利用亲和素-生物素免疫组织化学技术,研究了流行性乙型脑炎(简称乙脑)病毒抗原(P3,A2株)感染小鼠中枢神经系统后的定位。观察到乙脑病毒抗原分布具有一定选择性,主要集中在脑干、小脑、间脑和大脑皮质。病变分布与H-E常规病理染色一致。该方法敏感、特异。同时比较了兔免疫血清与乙脑单克隆抗体的应用效果。对组织化学冰冻切片漂浮法诊断病毒抗原进行了探讨。     

各种癌基因蛋白微波抗原修复免疫组织化学检测的研究

本研究对1555例各种肿瘤组织标本进行微波辐射(MWI)抗原修复免疫组织化学染色,检测了14种癌基因蛋白产物.并同时选择部分标本分别作冰冻切片,常规石蜡切片进行抗原修复与不修复的对比研究.结果,经MWI抗原修复后p53(PAb1801,PAb-240,PAb-420,DO-7),p185,Rb,C-fos,C-myc,Ki-67(R),EGF,EGFr,TGFβ和Bcl-2阳性检测率明显提高(P≤0 05),而p53(CM-1),PcNA p21和nm23/NDPK经MWI抗原修复与不修复结果一致,说明有部分抗原不用修复和其他处理.另一部分抗原仅表达在冰冻切片上,如Ki-67和Ki-1单克隆抗体,经修复也无阳性表达.本文中对消化、消化与抗原修复相结合及高压处理切片的IH染色也进行了对比研究,对他们处理切片存在的优缺点也一一作了详说.     

两种甲型肝炎病毒抗原快速检测方法的建立

目的建立两种甲型肝炎病毒抗原(HAV-Ag)检测试剂盒,并对其检测效果进行评价。方法生物素标记甲型肝炎病毒抗体(HAV-Ab)与辣根过氧化物酶标记亲和素联合应用建立甲型肝炎病毒抗原BA-ELISA检测法;同时使用辣根过氧化物酶标记HAV-Ab作放大系统建立双抗体夹心甲型肝炎病毒抗原ELISA检测试剂,对比两种检测方法的特异性、灵敏度及实际应用效果。结果用生物素标记甲型肝炎病毒抗体-辣根过氧化物酶标记亲和素作放大系统建立的甲型肝炎病毒抗原BA-ELISA检测法,较双抗体夹心ELISA检测方法灵敏度高1～2个稀释度;两种检测法均对10余种病毒无交叉,P/N值BA-ELISA检测法较高。结论甲型肝炎病毒抗原BA-ELISA检测法是一种灵敏度高,特异性好,方便快捷的检测方法,可广泛应用于甲型肝炎病毒研究及临床检测中。而甲型肝炎病毒抗原双抗体夹心ELISA检测法,检测灵敏度适中,操作简单,更适用于甲肝疫苗生产检定。     

应用微波辐射促进组织块浸银染色

目的：探索微波辐射对组织块浸银染色各步骤的作用及微波在其中的应用方法。方法：在组织块浸银染色过程中,对组织块的固定,浸银,还原等步骤进行不同强度的微波辐射处理。结果：微波辐射明显促进了浸银,还原作用,应用适宜强度的微波辐射染色效果比常规染色有更多的优点。结论：恰当地应用微波辐射缩短组织块浸银染色时程,切实可行。     

免疫聚合酶链反应技术的建立及其对甲脂蛋白的检测

免疫ＰＣＲ是一种新的具高敏感性的抗原检测技术,它类似传统的ＥＬＩＳＡ法,若与抗体连接的酶用ＤＮＡ片段代替,则该ＤＮＡ片段可用于ＰＣＲ扩增。因此,免疫ＰＣＲ有可能作为一种具高敏感性的检测手段用于临床早期诊断。     

盐酸酸化可改善核抗原免疫荧光染色

免疫荧光染色(immunofluorescent staining, IF)技术广泛用于细胞或组织内抗原定性、定量或定位检测。然而,依常规染色步骤操作,在有些胞核抗原的检测中很难得到令人满意的结果。有研究者采用盐酸酸化预处理用于细胞增殖标记物5-溴-2-脱氧尿嘧啶核苷(BrdU)的荧光染色并获得良好效果。但此方法是否也适用于其他类型的胞核抗原,尚不清楚。为系统全面分析盐酸酸化在胞核抗原免疫荧光染色中的作用,本文以成年C57BL小鼠主要嗅觉表皮(MOE)为材料,分别对Ki-67、5-甲基胞嘧啶(5mC)和5-羟甲基胞嘧啶(5hmC)三种不同类型的胞核抗原进行盐酸酸化处理免疫荧光染色。结果显示,当血清封闭和抗体浓度等条件一致时,室温下盐酸酸化2 h,Ki-67的染色效果最佳,而阴性对照与未酸化组均未出现阳性信号;同样经过盐酸处理2 h,5mC和5hmC染色也呈现较强的阳性信号。该研究表明,在一些胞核抗原免疫荧光染色中,使用盐酸酸化处理可显著提高染色效果。     

Microwave Irradiation for Rapid and Enhanced lmmunohistochemical Staining: Application to Skin Antigens

We describe a method in which microwave irradiation is used to reduce substantially the incubation time for immunoperoxidase staining of antigens in cryostat sections of pso-riatic skin. An incubation time of 5-9 min irradiation at 80 W generated similar or better staining intensity for all antibodies used compared to the standard methods using 30-60 min incubation at room temperature. Although we found that microwave irradiation could be used with all antibodies tested, independent of whether they recognized extracellular, membrane or cytoplasmic antigens in skin, the conditions needed to be optimized for each antibody.     

Electrochemical antigen-retrieval of formaldehyde fixed and paraffin-embeddedarchived leprosy skin biopsies

Summary While formaldehyde fixation preserves tissue morphology, it often hinders immunodetection of antigens in paraffin-embedded tissue because the antigens are masked. Antigen unmasking can be achieved with treatments such as microwave irradiation but they often lead to excessive tissue damage. Therefore, an electrochemical antigen-retrieval method (EAR) was devised in which an alternating electric current is passed through the tissue in a chamber containing an electrolyte buffer. The results obtained with this method were compared to those after microwave irradiation using archived samples of formaldehyde-fixed and paraffin-embedded lepromatous leprosy skin. The efficacy of the two unmasking procedures was assessed by the immunodetectability of several marker antigens using 24 antibodies. Fifteen antibodies that were directed against transmembrane proteins (CD), and the remaining 9 against cytokeratins 18.6 and 19, laminin, vimentin, S100a, BCG,Ulex europaeus lectin, PCNA, and P21^ras. Simple and double immunohistochemistry was performed using the universal ENVISION and LSAB + AP detection systems. After unmasking with the EAR method, immunoreactivity was clearly detected with 22 of the 24 antibodies in single labeling reactions. They include the critical antigens CD3 and CD4 for identifying the T lymphocyte lineages. In contrast, only 20 of the antibodies reacted after microwave irradiation. After double immunolabeling, immunoreactivity was quantitatively similar with both methods. However, the EAR unmasking produced a stronger labeling reaction. Thus, with double labeling immunohistochemistry, EAR made it possible to use higher antibody dilutions and shorter incubation times. Heat damage was also prevented. In conclusion, EAR treatment produces better staining results than microwave irradiation treatment.     

Repopulation of the mouse thymus after sublethal fission neutron irradiation. II. Sequential changes in the thymic microenvironment

The stromal cells of the thymus of sham-irradiated and sublethal fission neutron-irradiated CBA/H mice were analyzed with immunohistology, using monoclonal antibodies directed to I-A and H-2K antigens as well as specific determinants for cortical and medullary stromal elements. In the control thymuses, I-A expression in the thymus shows a reticular staining pattern in the cortex and a confluent staining pattern in the medulla. In contrast, H-2K expression is mainly confluently located in the medulla. Whole body irradiation with 2.5 Gy fission neutrons reduces within 24 hr the cortex to a rim of vacuolized "nurse cell-like" epithelial cells, largely depleted of lymphoid cells. The localization of I-A antigens changes in the cortex and I-A determinants are no longer associated with or localized on epithelial reticular cells. Medullary stromal cells, however, are more or less unaffected. A high rate of phagocytosis is observed during the first 3 days after irradiation. About 5 days after irradiation, the thymus becomes highly vascularized and lymphoid cells repopulate the cortex. The repopulation of the thymic cortex coincides with the appearance of a bright H-2K expression in the cortex which is associated with both stromal cells as well as lymphoid blasts. During the regeneration of the thymus, the thymic stromal architecture is restored before the expression of cell surface-associated reticular MHC staining patterns. The observed sequential changes in the thymic microenvironment are related to the lymphoid repopulation of the thymus.     

Immunoperoxidase staining of cervicovaginal smears after radiotherapy.

Cervicovaginal smears from 2 women with postirradiation dysplasia, 4 women with postirradiation squamous cell carcinoma of the cervix, 30 women with irradiation atypia and 5 healthy, nonirradiated women were stained immunohistochemically with six keratin antibodies. For four of the antibodies--CK19 (BA17), EMA, PKK-1 and CAM 5.2--squamous cells showing irradiation atypia, postirradiation dysplasia or postirradiation squamous cell carcinoma were more likely to stain positively than were nonirradiated squamous cells. For three of the antibodies in which multiple squamous cells stained positively, the proportion of squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma staining strongly was equal to or greater than the corresponding overall proportion for squamous cells showing irradiation atypia. This was statistically significant with only one antibody, PKK-1. No statistically significant differences were seen in staining of irradiated and nonirradiated squamous cells by MAK-6 and AE1:AE3. The data show that some keratin antigens are more often expressed in the irradiated groups and that there may be differences in the degree of antigen expression between squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma and squamous cells showing irradiation atypia.     

改进的免疫胶体铁细胞化学染色

本文以改进的方法制备了胶体铁。该胶体制备简单,稳定性高,易于抗体标记,在PH7.4时,胶体与抗体IgG稳定结合形成胶体抗体复合物的经以CM-Sephadex C-50代替AmberlightCG50进行标记物的纯化,方法可靠,所制备的胶体铁标记抗体用于免疫细胞化学染色,普鲁士蓝反应清晰地显示出标记抗体与相应抗原的特异性结合部位,与传统的免疫酶及免疫金银方法比较,具有敏感性高,背景干净,呈色鲜明等优点,结合免疫酶及免疫金银染色可用于抗原的双标及多标记。     

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling.

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.     

Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.     

Photobleaching of arterial autofluorescence for immunofluorescence applications

Immunohistochemical localization of low-level antigens in the arterial vasculature is complicated by the presence of complex molecules such as collagen, elastin, cholesterol, and fluorescent lipids that exhibit autofluorescence over a wide spectrum of wavelengths. UV irradiation of arterial vasculature has remained ineffective in preparing samples for immunofluorescent staining because of the recovery of the endogenous fluorescence within a short time following treatment. Therefore, we sought to further enhance the signal-to-noise ratio in arteries by optimizing the photobleaching of this tissue. We report here that the use of filtered sunlight significantly reduces arterial autofluorescence compared to standard UV shortwave and longwave irradiation and maintains multiple antigen epitopes suitable for immunohistochemical analysis. Using this method, we localized low-level laminin-5 isoform expression in situ, which was previously indistinguishable from endogenous autofluorescence.     

Using microwave irradiation in Marchi's method for demonstrating degenerated myelin

Conventional methods for histological preparation of degenerated myelin are time-consuming and difficult. The purpose of our study was to shorten the time required for the procedure and to obtain better quality results for light microscopic demonstration of degenerated myelin in the central and peripheral nervous systems by using microwave irradiation. Rat brain and sciatic nerve were used for the study. The middle cerebral artery was occluded and the sciatic nerve was cut to produce myelin degeneration. Marchi's method was used for staining degenerated myelin. Fixation for light microscopy that would take two days using the conventional procedure was completed in 16.5-18.5 min using microwave irradiation. While staining of degenerated myelin requires 10 days for the conventional Marchi method, we decreased it to 7 h for brain tissue and 1 h for sciatic nerve by using the microwave oven. Moreover, a better quality preparation was achieved in the groups stained under microwave irradiation than those prepared by the conventional method.