钨酸钠作为稳定剂的新的高灵敏HRP-TMB法——Ⅱ、电镜研究

本文研究了各种不同锇化步骤对TMB-ST反应产物的影响。结果发现:TMB-ST法结合DAB-Co后处理步骤用于电镜束路追踪时,具有反应产物电子密度很高,呈特征性的细杆状,组织超微结构保存良好的特点,且灵敏度较高。而未后处理的TMB-ST产物锇化后大多数只具有中等电子密度。此外,通过对以硝普钠、钼酸铵、柠檬酸镍离子络合物、钨酸钠作为稳定剂的各种TMB法的比较,我们首次提出了稳定剂是影响TMB产物电子密度的最重要因素的观点。     

钨酸钠做为稳定剂的新的高灵敏HRP-TMB法——Ⅰ、光镜研究

本文通过试管实验和在尾核—黑质投射模型上的比较研究,首次提出了一种以钨酸钠作为稳定剂的新的TMB成色法(TMB-ST法)用于HRP神经束路追踪术。与现确灵敏度最高的Mesulam的TMB法相比,TMB-ST法具有灵敏度更高,产物稳定,耐酒精、耐pH小于6.3的环境,无针状结晶和点状结晶,标记结构形态细腻,组织切片不皱缩等优点。此外,本文还对稳定剂在HRP-TMB组化反应中的作用作了较全面的讨论。     

钼酸铵固定TMB反应产物方法的探讨

钼酸铵能固定四甲基联苯胺(TMB)反应产物使之抗酒精的退色作用,已有报道。本文则分别探讨其耐酒精及高pH的作用。用辣根过氧化物酶(HRP)标记的切片经TMB温育后用钼酸铵处理,再置入pH3.3的酒精30分钟,可见酒精对钼酸铵固定后的TMB反应产物无退色作用;若放入pH7.4的磷缓则明显退色,但比未经钼酸铵处理者退色慢。     

大鼠垂体后叶中多巴胺能纤维的起源

成年Sprague-Dawley大鼠在戊巴比妥钠麻醉下,于垂体后叶直视下注入CT-HRP和WGAHRP混合液0.5μl,12小时后,经侧脑室注入秋水仙素120μg。再存活12～24小时后,用3.5％多聚甲醛经升主动脉灌注固定。连续冰冻切片,厚40μm。TMB成色后,再用DAB-硫酸镍胺方法加强。用抗酪胺酸羟化酶抗体孵育48小时后,用ABC方法呈色。镜下见有3种不同的神经元:1.单纯紫蓝色颗粒的神经元;2.单纯棕黄色标记的神经元;3.     

HRP—TMB反应中醋酸缓冲液的pH值与反应敏感性、组织超微结构保存的关系

为了探索HRP—TMB电镜技术中既能保持TMB反应敏感性高的优点又可较好地保存组织超微结构象,本文将WGA—HRP注入大白鼠红核及其周围,通过用不同pH值的醋缓配的反应液(pH3.3,4.0,4.2,4.4,4.6,4.8,5.2,5.6)后,在光镜及电镜下观察了下橄榄核区标记终末的密度及组织超微结构像。生现醋缓pH为4.0及3.3时,标记终末密度相似,或4.0时多于3.3,但4.0的组织像较3.3者有明显的改善。当pH值进一步提高时,尽管电镜下组织像逐渐改善,但标记的密度却逐步下降,至5.6时,几无阳性标记。另外,pH4.0的材料中,TMB反应产物变小,其跨膜现象也较3.3者少见。故作者认为,选用pH值为4.0的醋缓液较为合适。     

一种较理想的用于ELISA的辣根过氧化酶底物

比较了辣根过氧化酶(HRP)的三种还原底物,邻苯二胺(O-PD)、2,2′-连氮-双〔3-乙基苯并噻唑啉-6-磺酸盐〕(ABTS)及3,3′,5,5′-四甲基联苯胺(TMB)的动力学,发现(1)不论以游离 HRP、HRP-IgG 或 HEP-Fab′为酶制剂,其 Km 均以 TMB 为最小;(2)TMB 所需的 H_2O_2最适浓度最低;(3)以 TMB 为底物时,酶活力受 pH 的影响最小;(4)酶活力测定的灵敏度最高;(5)蓝色的氧化产物可用于酶标定位,而加酸停止反应后的黄色产物又可用于酶标免疫测定的定量比色。再加上三种化合物中,仅 TMB 为非致癌性,故 TMB 是一个比较理想的 ELISA 的 HRP 底物。     

麦芽凝集素辣根过氧化物酶、类霍乱原结合辣根过氧化物酶及辣根过氧化物酶—四甲基联苯胺法成色脑切片的保存时间探讨

目前,辣根过氧化物酶(HRP)法已被推广和改进,在HRP束路追踪技术中,四甲基联苯胺(TMB)因其灵敏度高又无致癌作用而被广泛采用.一般认为TMB反应产物易受高pH、酒精、温度、紫外线照射等因素影响而退色.TMB呈色时加钼酸铵再稳定,有利于切片的保存.能保存多久.其消退程度如何,采用麦芽凝集素辣根过氧化物酶(WGA-HRP)、类霍乱原结合辣根过氧化物酶(CB-HRP)及HRP(Sigma VI)法制作的切片,保存时间上有无明显差别是本文研究的目的所在.     

金属离子诱导人单核细胞凋亡并释放肿瘤坏死因子的实验研究

目的 探讨人工关节磨损产物金属离子钴(Co2 )、铬(Cr3 )对人工关节无菌性松动的影响.方法 将金属离子Co2 、Cr3 分别与人单核细胞共培养,分别于12、24、48小时取细胞上清液,用ELISA法检测上清液中肿瘤坏死因子(TNF-α)含量,并测定细胞悬液中细胞凋亡情况.结果 Co2 、Cr3 均能刺激人单核细胞产生TNF-α且TNF-α量随时间上升,一定时间后达到峰值,随后缓慢下降.而随时间发展,人单核细胞凋亡率逐步升高.结论 金属离子Co2 、Cr3 能通过刺激人单核细胞合成和释放肿瘤坏死因子,诱发人单核细胞凋亡从而引起假体周围骨溶解,进而引起假体松动.     

免疫电镜技术运用过程中电子染色方法的选用

目的探究免疫电镜不同染色方法对免疫组化阳性实验结果影响的关系。方法组织切片经常规免疫组化（雌激素受体GPR30）并行DAB-硫酸镍铵显色后进行电镜切片，然后分为双氧铀-柠檬酸铅双染、双氧铀单染与未染色3组，以便对不同电子染色结果进行比较。结果GPR30免疫阳性产物位于细胞核外的膜性结构上，在铀-铅双染组显示很高的电子密度，但是背景染色也很深，在铀单染组的反差比较好，而未染色组的反差更好。结论免疫电镜技术中针对不同的免疫阳性反应选用不同的电子染色方法，有利于阳性结果的判断与鉴别。     

用铈作捕捉剂检测横纹肌细胞Ca^2＋—ATP酶活性

本文介绍了一种新捕捉剂铈在横纹肌Ca~(2+)-ATP酶电镜细胞化学中的应用。与铅法相比,以铈为捕捉剂的电镜细胞化学方法有反应稳定、非特异性反应少、反应产物细而均匀等优点。将孵育液pH由铅法的8.8～9.0改为7.2～7.4,能显示肌膜Ca~(2+)-ATP酶的活性。     

Modified cerium-based and Gomori-based cerium methods for light microscopic phosphatase histochemistry: the cerium-perhydroxide-diaminobenzidine-nickel (Ce-H2O2-DAB-Ni and Ce/Ce-H2O2-DAB-Ni) two-step procedures.

Some modified cerium-based and Gomori-based cerium methods for the demonstration of phosphatase activity in cryostat sections were described. Dextrane as stabilizing agent was added to the incubation media for ATPase, 5'-Nase, and TPPase. The oxidation of the CeIII-phosphate primary reaction product in a separate step by H2O2 before the DAB incubation yielded an increase of the intensity of the DAB-based visualization reaction (Ce-H2O2-DAB-Ni two step method). The sensitivity of the histochemical enzyme reaction was remarkably increased if CeIII-ions were employed as amplifying agent (Ce/Ce-H2O2-DAB-Ni two-step method). A new suitable DAB medium consisting of 0.015% DAB, 2.0% Ni-sulphate, 15% methanol, and 0.005% H2O2 in 0.1 mol/l acetate buffer, pH = 5.2, was used. The disadvantage of diffuse background staining has been overcome by addition of 15% methanol to the DAB solution. Electrovalently bound CeIII (cerophilia) was removed by treatment of the incubated sections with CeIII-citrate (CeIII-complexation). In addition, a novel membrane floating incubation for sections is proposed. At present, the modified procedures are some of the most sensitive modes for the demonstration of phosphatases and improve the earlier described cerium-DAB one-step technique (Halbhuber et al. 1988b).     

The use of tetramethylbenzidine for solid phase immunoassays

A rapid, sensitive, solid-phase immunoassay, using horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine, was found to be more sensitive and the colour developed was more stable compared with HRPO using 4-chloro-1-naphthol or diaminobenzidine. Assay sensitivity was equal to or greater than that obtained with the alkaline phosphatase reaction. The essential step was to incubate filters in 1% dextran sulphate for 10 min in a pH 5 (10 mM citrate-EDTA) buffer before reacting with TMB (0.05 mg/ml in the same buffer).     

在同一神经元内同时显示Fos－蛋白、酪氨酸羟化酶和HRP逆行标记的三重标记技术

本文介绍了一种能在同一神经元内同时显示ＨＲＰ逆行标记以及Ｆｏｓ和某类神经递质或相关酶（例如ＴＨ）的三重标记技术。首先将ＨＲＰ注入脑内某一核团（如中央杏仁核），动物存活４８ｈ后，再向胃内导入１％福尔马林２ｍｌ，以造成对胃的伤害性刺激，２ｈ后将动物处死。脑的切片（如本文的延髓切片）先用四甲基联苯胺－钨酸钠（ＴＭＢ－ＳＴ）法进行ＨＲＰ呈色反应，后用二氨基联苯胺（ＤＡＢ）和氯化钴作加强；然后按ＡＢＣ法进行Ｆｏｓ免疫组织化学反应，继之按ＰＡＰ法进行ＴＨ免疫组织化学反应。光镜下在延髓切片观察到７种标记神经元，即ＴＨ－、ＨＲＰ－或Ｆｏｓ－单标记神经元，ＴＨ和ＨＲＰ－、ＴＨ和Ｆｏｓ－或ＨＲＰ和Ｆｏｓ的双标记神经元以及ＴＨ和ＨＲＰ和Ｆｏｓ的三标记神经元。该方法为研究同时显示中枢神经元的功能活动状态、传出投射及所含神经递质提供了一个强有力的工具。     

Employment of merocyanine 540 fluorescence to form diaminobenzidine (DAB) oxidation product: a photoconversion method for the visualization of erythrocyte membrane fluidity for light and electron microscopy

Intact native red blood cells (RBC) and treated RBC preparations were labelled with MC 540 and irradiated in the presence of diaminobenzidine (DAB). The polymerized diaminobenzidine reaction product is permanently stable in comparison with the labile fluorescence labelling. The brownish stained DAB polymerization product (DAB brown) and osmium black (after conversion of DAB brown with OsO4) allow the densitometrical determination with the light microscope. The latter product can be directly observed in the electron microscope. A direct correlation exists between the fluorescence intensity and the polymerized diaminobenzidine staining. It can be deduced that the enhancement of the DAB mediated contrast is reflecting an increased fluidity of the red cell membrane. The reaction was successful with all red cell preparations tested. This method is also suitable for the determination of fluidity changes in other cell membranes.     

A safer and more sensitive substitute for diamino-benzidine in the light microscopic demonstration of retrograde and anterograde axonal transport of HRP

The commonly used benzidine derivatives such as diaminobenzidine, o-dianisidine and benzidine dihydrochloride are apparently carcinogenic. Availabel information, however, indicates that tetramethyl benzidine (TMB) is safer in this regard, and we have therefore developed a procedure in which TMB is used as a substrate for the detection of intra-axonal transport of HRP. The TMB procedure is simple, reliable and does not produce crystals if properly executed. The staining procedure, which takes about 15 min, produces a blue reaction product which is easily detected in bright-field microscopy.     

Endogenous peroxidase-like activity in the feline dorsal column nuclei and spinal cord

Summary Endogenous peroxidase-like activity was investigated with a combined light and electron microscopical technique in 15 cats. The lateral cervical nucleus, the dorsal column nuclei, and segments C6 and L5 of the spinal cord were incubated with diaminobenzidine-tetrahydrochloride (DAB) or tetramethylbenzidine (TMB). After histochemical reaction with DAB a considerable amount of activity was found in nerve cells, astrocytes and pericytes. The neuronal labelling was mainly located in mitochondria of axon terminals and in dendrites whereas the astrocytic and pericytic activity was found in cytoplasmic dense bodies. The quantity of stained structures differed considerably between the animals. In TMB reacted tissue endogenous peroxidase-like activity was only sparsely seen. It was found mainly in frozen sections, in which the neuropil and perivascular structures sometimes contained granules and irregular filaments. The significance of the findings is discussed in relation to observations in tracer studies using horseradish peroxidase.