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1.
应用免疫组织化学碱性磷酸酶标A蛋白(PAAP)技术在光镜水平研究中国树鼠句伏隔核内促肾上腺皮质激素释放激素(CRF)能神经元的形态和分布特点。结果显示,该核内CRF免疫反应阳性神经元胞体多数呈多边形、圆形或卵圆形,梭形极少;直径多数为13-19um,少数<13um;胞质免疫反应强度不等。对左右侧伏隔核内CRF免疫反应阳性神经元数目、胞体大小、形态和免疫反应强度进行分析,除免疫反应强阳性神经元计数项(P<001)外,其他项都无显著意义。CRF免疫反应阳性神经元在伏隔核内分布不均,主要位于该核的前半段背侧区,核芯区较少 相似文献
2.
本文研究了免疫组织化学方法中碱性磷酸酶(AP)的显色系统。本文以人扁桃体的石蜡切片为材料,以CD20为标记,二抗为生物素标记的兔抗鼠抗体,选择了5种萘酚磷酸(naphtholAS-OLphosphate,naphtholAS-GRphosphate,naphtholAS-TRphosphate,naphtholAS-MXphosphate和naphtholAS-BIphosphate)与9种重氮化合物(fastredTRsalt,fastblackKsalt,fastblueBBsalt,fastbrownRRsalt,fastscarletRsalt,fastbluesaltBN,fastblueB,fastdarkblueRsalt,fastredvioletLBsalt)以及新品红为AP显色底物,相互配伍,根据形成的一系列显色反应,比较研究了不同的底物对。研究结果表明:在5种萘酚磷酸中,naphtholAS-OLphosphate具有良好的显色敏感性,当一抗为1∶1000时,与常用naphtholAS-BIphosphate一样,具有较强的显色反应;该底物还具较好的配伍性,能与新品红和FastBlue 相似文献
3.
光系统Ⅱ核心天线CP43的纯化及性质 总被引:7,自引:0,他引:7
菠菜放氧的PSII核心复合物经0.8mol/LTris-HCl(pH8.0)洗涤后,用温和的非离子去垢剂DM和高浓度的LiClO4增溶,再经DEAE-Toyopearl-650S离子交换柱层析分离,可得到PSII核心天线43kD叶绿素a结合蛋白(CP43)。SDS-PAGE显示一条43kD蛋白质带。根据Arnon法和Markewll法的结果表明,每个蛋白质分子结合20~21个分子的叶绿素a。室温条 相似文献
4.
小分子G蛋白Ras超家庭成员都是通过与鸟苷酸交换因子(guanine nucleotide exchange factors,GEFs)结合而被活化,GEFs能增加这类小分子G蛋白对GTP的摄取,并使之形成有活性的GTP结合构象。Rap1是Ras样的小分子G蛋白。迄今为止,已发现G3G、CalDAG-GEF1、Epac、cAM-GEF1、cAMP-GEFⅡ、nRapGEP、GFR可特异的活化Rap 相似文献
5.
美洲拟鲽抗冻肽基因在E.coli中的表达 总被引:10,自引:0,他引:10
动物抗冻肽(AFP)以降低动物体液冰点而使其能在寒冷的环境中生活,因而AFP在防寒抗冻方面有较大的潜力。根据美洲拟鲽AFP基因的cDNA序列,人工合成了AFP及春含前导序列的proAFP的cDNA,并构建成PAFP及PPAFP原核表达载体。由于AFP表达量少且蛋白分子很小,用SDS-蛋白电泳难以检测。为了提高检测的灵敏度,把报道基因CAT的CDNA按读码框连接到AFP的3'末端,构建成融合基因表达 相似文献
6.
光动力复合藻胆蛋白及其分子内能量传递现象 总被引:2,自引:0,他引:2
通过杂双官能团偶联试剂,3-(2-吡啶联疏基)丙酸N-羟基琥珀酰亚胺酯(SPD),我们合成了R-藻红蛋白(R-PE)与变藻蓝蛋白(APC)的共轭复合物。这种光动力复合藻胆蛋白具有一些特别的光物理性质,如很大的stokes位移(约170um,490nm激发,665nm发出荧光)、高效的分子内能量传递效率等。复合物中R—PE与APC的摩尔比为14,且R—PE和APC在复合物中保持了它们各自的光谱性质。通过荧光发射光谱,我们观察到了这种复合藻胆蛋白的分子内能量传递现象,计算表明从R—PE到APC的分子内能量传递效率为65%。二硫苏糖醇(DTT)对复合物中联结R—PE与APC间的脂族二硫桥键的还原导致分子内能量传递的阻断这一现象进一步证实了复合物中存在分子内能量传递现象。根据Forster能量转移机理,计算得出给体与受体发色团间距离为72A,这一距离与两种蛋白的大小是基本相符的。 相似文献
7.
菠菜放氧的光系统Ⅱ(PSⅡ)核心复合物经0.8mol/L Tris(pH8.0)洗涤后,用温和的非离子去垢剂DM和高浓度的LiClO4增溶,再经DEAE-Toyopearl-650S离子交换柱层析分离,可得到PSⅡ天线组分中的叶绿素α/b结合蛋白(CP29)。SDS-PAGE显示一条30kD蛋白质带。根据Arnon法和Markwell法的结果表明,每个蛋白质分子结合有7~8个分子的叶绿素α和2~3 相似文献
8.
本文探讨了具有肿瘤抑制功能的cDNA克隆P14-6(即人白细胞介素6核转录因子NF-IL6的3’非翻译区)的RNA转录物与回复相关蛋白BNF的相互作用,发现该RNA与BNF的相互作用位点为其3’侧的富U序列内的1个24核苷酸片段;并发现BNF系一群蛋白质,它们可能先相互结合成1个蛋白质复合物,然后再与RNA位点作用.其中可能只有1个蛋白质(R62)直接与该RNA结合。 相似文献
9.
应用荧光原位杂交和RFLP标记检测多小穗小麦新种质10—A中的黑麦染 … 总被引:4,自引:0,他引:4
利用APAGE,荧光原位杂交技术和RFLP标记,对导入黑麦(SecalecerealeL.)多小穗等性状创制的小麦新种质10-A进行了分子标记检测,APAGE分析发现,10-A与其他1RS/1BL易位系一样,含有1RS的醇溶蛋白标记位点Gld1B3。以黑麦基因组总DNA作探针,用中国春(Triticumaestiumcv.ChineseSpring)基因组DNA作封阻,与10-A根尖细胞有丝分裂染 相似文献
10.
为探讨八肽胆囊收缩素(CCk-8)和阿片肽相互作用的分子机理,利用抗体免疫沉淀技术研究了CCK-8与NDAP(k阿片受体激动剂)对大鼠脑(去皮层和小脑)和脊髓背柱组织Fos蛋白的影响。结果表明,0.1μmol/LCCK-8可显著刺激脑和脊髓组织中Fos蛋白增加(分别是对照组的3.8倍和3.6倍)。相同浓度的NDAP对Fos蛋白的生成亦有一定的诱导作用,分别是对照组的2.7倍和2.6倍。CCK-8和NDAP共同处理组织,Fos蛋白生成水平相似(脑)或高于(脊髓)CCK~-8单独诱导的水平。结果表明,CCK-8和NDAP均可直接诱导大鼠脑和脊髓组织c-fos的表达,它们对c-fos表达的相互作用在脑和脊髓中呈现不同的模式。 相似文献
11.
G Mechtersheimer P M?ller F Momburg G Moldenhauer K Schwechheimer 《The journal of histochemistry and cytochemistry》1986,34(3):399-402
A sensitive immunoenzymatic double-staining technique is presented for the simultaneous visualization of lectin-binding sites and antigenic structures detected by monoclonal antibodies. The lectin is demonstrated by an extended unlabeled peroxidase-antiperoxidase (PAP) technique and the monoclonal antibody by an alkaline phosphatase-antialkaline phosphatase (APAAP) method, which corresponds to the standard PAP technique. 3-amino-9-ethylcarbazole (AEC) and fast blue BB salt serve as substrates for the peroxidase and the alkaline phosphatase, respectively. The antisera and the enzyme complexes raised in different animals enable the performance of three parallel incubation steps. The staining procedure requires three and a half hours altogether. This method proved to be highly discriminative and rather insensitive to interference by various artifacts. 相似文献
12.
A highly sensitive assay method for human placental alkaline phosphatase involving a monoclonal antibody bound to a paper disk 总被引:3,自引:0,他引:3
K Hirano Y Iiizumi Y Hayashi T Tanaka M Sugiura K Hayashi Z D Lu S Iino 《Analytical biochemistry》1986,154(2):624-631
A monoclonal antibody which is specific for human placental alkaline phosphatase and does not cross-react at all with intestinal alkaline phosphatase was prepared, and a procedure for the determination of placental alkaline phosphatase activity in serum was developed involving this monoclonal antibody bound to a paper disk. The minimum amount of placental alkaline phosphatase detectable by this method is 0.0025 King-Armstrong unit. Good correlation with the heat-treatment method was obtained. Therefore this proposed method can be used as a routine clinical test for the determination of serum placental alkaline phosphatase. 相似文献
13.
Visualization of antigenic proteins on Western blots 总被引:31,自引:0,他引:31
A new technique for the detection of antibodies bound to proteins blotted onto nitrocellulose paper was developed. The method is rapid, sensitive, and does not require radioactive probes. Proteins transferred to nitrocellulose paper are first reacted with primary antibody followed by reaction with an alkaline phosphatase conjugated second antibody. The phosphatase activity is then visualized using an agar gel impregnated with the histochemical phosphatase stain 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (J. P. Horwitz, J. Chua, M. Noel, J. T. Donatti, and J. Freisler (1966) J. Med. Chem. 9, 447; Sigma Chemical Co., Technical bulletin No. 710-EP (1978]. Antigen-antibody complexes give rise to sharp, permanent blue stained bands both on the nitrocellulose paper and in the agar overlay gel. This procedure allows detection of bands containing less than 20 ng of protein. 相似文献
14.
R P Linke 《The journal of histochemistry and cytochemistry》1984,32(3):322-328
Monoclonal antibodies against amyloid fibril protein AA were produced by cell fusion of murine P3 X 63-Ag8.653 myeloma cells with spleen cells of immunized Balb/c mice. To increase immunogenicity, protein AA was coupled to horseradish peroxidase (HRP) or human high molecular weight kininogen (HMWK). Using micro-ELISA (enzyme-linked immunosorbent essay) seven hybridoma cell lines secreting antibodies that specifically bind to protein AA have been selected and cloned. When applied to formalin-fixed paraffin sections of a variety of different amyloid types using immunoperoxidase methods, five monoclonal antibodies bound specifically and strongly to amyloid only of the AA type. Since a series of different AA-amyloids could be stained, these reagents may be used to routinely diagnose AA-amyloidosis in tissue sections. A monoclonal antibody against HRP has also been produced that has been utilized to develop a monoclonal peroxidase-antiperoxidases (PAP) complex. When three immunoperoxidase methods were compared, the sensitivity of a conventional rat PAP was comparable to the monoclonal PAP complex, but the latter was easier to handle. Both methods were more sensitive than the indirect immunoperoxidase technique. 相似文献
15.
A. H. N. Hopman Sandra Claessen Ernst J. M. Speel 《Histochemistry and cell biology》1997,108(4-5):291-298
We describe the brightfield microscopical detection of multiple DNA target sequences in cell and tissue preparations. For
this purpose, chromosome-specific DNA probes labelled with biotin, digoxigenin or fluorescein were simultaneously hybridised
and detected by enzyme cytochemistry using two horseradish peroxidase (PO) reactions and one alkaline phosphatase (APase)
reaction. For triple-colour detection on single cell preparations, the combination of the enzyme precipitates PO/diaminobenzidine
(DAB, brown colour), APase/fast red (FR, red colour) and PO/tetramethylbenzidine (TMB, green colour) resulted in an accurate
detection of DNA targets. Embedding of the preparations in a thin cross-linked protein layer further stabilised the enzyme
reaction products. For in situ hybridisation on tissue sections, however, this detection procedure showed some limitations
with respect to both the stability of the APase/FR and PO/TMB precipitates, and the sequence of immunochemical layers in multiple-target
procedures. For this reason, the APase/FR reaction was replaced by the APase/new fuchsin (NF, red colour) reaction and the
washing steps after the PO/TMB reaction were restricted to the use of phosphate buffer pH 6.0. Furthermore, to improve the
efficiency of the ISH reaction, APase/NF was applied in an avidin-biotin complex detection system and, to avoid target shielding
in the triple-target ISH, the third primary antibody was applied prior to the second enzyme cytochemical reaction. These adaptations
resulted in stable, well contrasting brown, red and green coloured precipitates. After quick haematoxylin counterstaining,
the tissue preparations were directly mounted in phosphate buffer and, optionally, embedded in the cross-linked protein layer.
Accepted: 27 June 1997 相似文献
16.
Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue-specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non-specific background staining in pathological liver specimens. We compared peroxidase-anti-peroxidase, alkaline phosphatase-anti-alkaline phosphatase (PAP/APAP), labelled-avidin-biotin (LAB/LAB) and digoxigenin-anti-digoxigenin (dig-a-dig/PAP) techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP-technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig-a-dig/PAP protocol. In contrast to the dig-a-dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue. 相似文献
17.
Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C. 总被引:3,自引:2,他引:1
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The subcellular distribution of alkaline phosphatase and penicillinase was determined by double labeling frozen thin sections of Bacillus licheniformis 749/C with colloidal gold-immunoglobulin G (IgG). Antipenicillinase and anti-alkaline phosphatase antibodies were used to prepare complexes with 5- and 15-nm colloidal gold particles, respectively. The character of the labeling of membrane-bound alkaline phosphatase and penicillinase was different: the immunolabels for alkaline phosphatase (15-nm particles) were bound to a few sites at the inner surface of the plasma membrane, and the gold particles formed clusters of various sizes at the binding sites; the immunolabels for penicillinase (5-nm particles), on the other hand, were bound to the plasma membrane in a dispersed and random fashion. In the cytoplasm, immunolabels for both proteins were distributed randomly, and the character of their binding was similar. The labeling was specific: pretreating the frozen thin sections with different concentrations of anti-alkaline phosphatase or penicillinase blocked the binding of the immunolabel prepared with the same antibody. Binding could be fully blocked by pretreatment with 800 micrograms of either antibody per ml. 相似文献
18.
Efficiency and sensitivity of indirect immunoperoxidase methods 总被引:3,自引:0,他引:3
The peroxidase-antiperoxidase (PAP) complex method has repeatedly been claimed to be more sensitive and antibody efficient than the indirect peroxidase labeled antibody method. However, most studies comparing these methods used tissue sections as the test material. However, test systems with known amounts of antigen will allow more reliable comparison of these methods and quantitative evaluation of method sensitivity. We therefore compared the antibody efficiency and sensitivity of these methods for the detection of human chorionic gonadotropin in an enzyme linked immunosorbent assay (ELISA), an antigen spot test (AST) and tissue sections of choriocarcinoma. In the PAP technique rabbit PAP and goat anti-rabbit antibody were applied. The same antibody was peroxidase-labeled with the periodate technique and used in the labeled antibody method. In the ELISA the PAP method resulted in slightly higher antibody efficiency than the labeled antibody method. At low primary antibody dilutions the intensity of the reaction decreased with the PAP method but remained high with the labeled antibody method, in the ELISA as well as on tissue sections. In the AST the labeled antibody method and the PAP method appeared to be equally sensitive. 相似文献
19.
C. M. Van Der Loos J. J. Van Den Oord P. K. Das H. J. Houthoff 《The Histochemical journal》1988,20(8):409-413
Summary An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green-and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes. 相似文献
20.
Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 1:2,400 of the primary antibody as compared to 1:800 for the PAP technique. 相似文献