赖夫生鞭毛染色法的改进*

赖夫生染色法是细菌鞭毛染色的一种常用方法。最近发现了延长赖夫生染剂使用寿命及改进染色效果的方法 ,并在此基础上进一步论证了乙醇在染色过程中所起的作用。     

提高网状纤维染色质量的技术探讨

网状纤维染色在病理工作中是一种最常用的特殊染色方法,它可以用来显示病变组织网状支架的破坏情况,对于判断病变的性质、程度及其发展与转归具有重要意义.尤其在肿瘤病理诊断中,对于鉴别来源于上皮组织和间叶组织的恶性肿瘤具有重要价值.网状纤维染色方法很多,但常用的方法是Gordon-Sweet氏法.该法是一种嗜银染色法,易受多种因素的影响,在染色过程中若操作不当,极易造成染色效果不佳.本文对此法进行了改良,并对其染色机理进行了探讨,同时对染色过程中易出现的问题,分析其原因,提出了具体解决办法,使染色质量明显提高.     

介绍一种改良的伊红染色液

在常规HE染色过程中,要求切片核浆对比要分明,细胞浆染色质量的好坏亦至关重要。伊红是细胞浆常用的染色剂,其配制方法较多,多用水溶性伊红染色液,在染色过程中发现水溶性伊红染色液易出现着色过淡、核浆共染及染色后的切片置一段时间后易褪色等现象,给观察切片的组织形态带来一定困难。我们根据伊红的化学性质和染色理论,结合实际应用情况,对传统伊红染液的配制方法进行了一些改良,收到了理想的染色效果。     

核酸染料Genefinder使用方法的比较

目的:使用核酸染料Genefinder检测琼脂糖凝胶中的核酸,通过比较预染样品法、胶内染色法和后染法三种染色方法的染色效果,了解该染料的染色特性,以期找到性能稳定,染色效果好的染色方法.方法:在琼脂糖凝胶电泳中,以不同的染色方法使用核酸染料Genefinder进行染色,对染色结果进行比较分析.结果:使用电泳后染色方法染色效果较好,胶内染色法次之,预染样品法效果最差.结论:核酸染料Genefinder会干扰DNA的迁移效率,因此,使用Genefinder进行电泳后染色是一种较好的染色方法.     

超薄切片染色后一种简易快速的洗涤法

超薄切片在电镜观察之前染色这一关是比较麻烦,也是比较重要的一关。现在LKB公司虽然已经生产出一种超薄切片自动染色器,每次同时能染色25个铜网,给超薄切片染色带来了很大的方便。但是该仪器价格昂贵(近一万美元),很不经济。因此目前大多数工作者仍以传统的方法进行染色,即液滴染色法。但用这种传统的方法染色是很麻烦的,尤其是在染色后     

蛋白质组学实验中聚丙烯酰胺凝胶的考马斯亮蓝染色

在蛋白质组学实验中,蛋白质经聚丙烯酰胺凝胶电泳后的染色是一个十分重要的环节。鉴于其良好的质谱兼容性和操作上的便捷,考马斯亮蓝染色是目前众多实验室中最常用的染色方法。本文介绍了四种各具特色的考马斯亮蓝染色方法。     

沉积物中活体底栖有孔虫的虎红染色方法比较研究

在有孔虫生态学研究中,区分底栖有孔虫活体与死亡壳体多使用虎红染色方法,但关于具体的染色时长、处理方式等方法描述比较简单,如仅有"采用虎红酒精染色",其具体方法常语焉不详,影响了研究结果比较的可靠性。为探求不同染色方法到底对实验结果产生多大偏差、有多少影响,本文利用经钙黄绿素培养的潮间带底栖有孔虫进行虎红酒精溶液方法对底栖有孔虫进行染色实验,结合活体有孔虫壳体生活时吸收的钙黄绿素能产生荧光效应,评估了不同保存条件下染色溶液的浓度、染色时间等处理方法下的染色效果。研究结果显示,2‰浓度的虎红酒精溶液适合区分活体与死亡底栖有孔虫壳体的染色要求,可以识别97%的活体底栖有孔虫;活体底栖有孔虫可以从一两个房室到多个房室被显著染色、呈现鲜艳的玫瑰红色,且这些被染色部分可以分布在早期、中期和后期房室。而实验保存处理方式以采集样品后立刻进行染色为佳,若条件限制则可使用纯酒精浸泡或冷冻保存处理后,尽快进行染色。染色时间一般在24小时以上较为合适,但对较长时间保存的样品则建议增加染色时长——两天以上,以达到染色效果。比较而言,酒精浸泡或冷冻保存比福尔马林浸泡样品具有较好的染色效果。     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

网状纤维染色技术改进

目的探讨改进网状纤维染色氨银液配制方法与改进染色方法的可行性。方法改进网状纤维染色中氨银液的配制方法和染色方法,与传统染色方法比较其染色效果与脱片率。结果改进网状纤维染色方法切片背景清洁度、网状纤维清晰度、网状纤维与胶原纤维对比度3项指标的得分均显著高于传统方法;改进网状纤维染色方法切片脱片率低于传统方法。结论改进网状纤维染色氨银液配制方法、改进网状纤维染色方法,可提高氨银液配制的成功率,延长氨银液有效期,提高网状纤维的染色质量,减少网状纤维染色的脱片率。     

植物树脂半薄切片染色方法的改进

以双子叶植物棉花的幼根、幼茎、叶、胚珠和单子叶植物玉米茎为实验材料,制作树脂半薄切片,对样品染色方法等进行了改进,将蕃红-固绿染色、苏木精-伊红染色、氯化铁-苏木精染色方法系统应用于植物树脂半薄切片制备中,获得了结构完整、染色清晰的棉花各器官和玉米茎半薄切片,建立了一套适合于植物树脂半薄切片制作的染色方法。     

Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain

In the current protocol, we describe the Congo red staining method and a method for separately quantifying vascular and parenchymal amyloid deposits in brain tissue sections. Congo red staining detects amyloid deposits in brain tissue of amyloid precursor protein transgenic mice and human Alzheimer's tissue. It detects compacted amyloid in a beta-sheet secondary structure and labels amyloid in both the brain parenchyma (amyloid plaques) and blood vessels. Congophilic amyloid in blood vessels is called cerebral amyloid angiopathy (CAA). To date, analysis of CAA has largely used a severity rating scale, including both qualitative and quantitative characteristics. Here, we describe a simple method for quantifying total Congophilic staining and resolution of this staining into the parenchymal and vascular components based on morphological criteria. It is becoming increasingly important to separately quantify various components of the Alzheimer's pathology, given the advancement of amyloid-lowering therapies into clinical trials. The entire procedure for the Congo red staining can be performed at room temperature (20-25 degrees C) in a fume hood. The staining protocol should take 1 h 30 min including time for coverslipping slides. Time required for image analysis depends greatly on the number of samples being analyzed and the software being used. In our hands, 30 images can be collected per hour and quantified in a further 2 h.     

Rapid method for distinction of gram-negative from gram-positive bacteria

Summary A rapid method for distinction between gram-negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram-positive and 55 gram-negative bacterial strains. The method proved reliable with one exception only, a Bacillus macerans strain. That strain was definately gram-negative on staining. Other Bacillus strains were proved gram-positive by the test, even those being gram-negative on staining.     

A comparative histochemical and immunocytochemical study on the secretory material in the subcommissural organ of Rana temporaria L

The secretory material within the cells of the subcommissural organ (SCO) of Rana temporaria has been studied histochemically by application of a classical staining method and immunocytochemically by use of an antiserum (gift of Prof. Sterba, Leipzig) raised against bovine liquor fibre (LF) material, the LF being the secretory product of the SCO. The immunocytochemical method appears to be more specific and more sensitive than the histochemical staining method. The LF can also be visualized immunocytochemically; the fibre reacting equally positively from the SCO until the spinal cord's end. A significant positive linear correlation exists both between the amounts of histochemically and of immunocytochemically stained material and between their concentrations. The suitability of the immunocytochemical staining method for quantification of the secretory material within the cells of the SCO is discussed.     

基于MTT染色法对葡萄生单轴霉孢子囊存活力的检测

本文利用MTT染色法和点接生物测定法对不同温度干燥处理36h后的葡萄生单轴霉孢子囊存活力和致病力进行了检测,并应用单因素试验和正交试验对其孢子囊进行了MTT染色条件的优化。结果表明：葡萄生单轴霉孢子囊MTT染色的最佳条件为温度36℃、MTT浓度 0.05%、染色48h,孢子囊染色率可达83.0%。20℃恒温干燥处理36h显著提高了孢子囊存活力,葡萄生单轴霉孢子囊的蓝色染色率为79.3%,叶片点接发病率为98.9%,显著高于对照的蓝色染色率52.0%和发病率62.7%。MTT染色得到的孢子囊蓝色染色率与点接生物测定法得出的发病率存在很好的线性关系y=1.276 1x-1.939 1,R²=0.996 1,孢子囊的蓝色染色率与叶片点接发病率呈正相关。本研究表明MTT染色法可以用于葡萄生单轴霉孢子囊存活力的快速和准确检测。     

嗜酸粒细胞过氧化物酶快速染色的研究

目的建立嗜酸粒细胞过氧化物酶快速染色方法,完善包括嗜酸粒细胞脱颗粒或中性粒细胞粗颗粒等各种情况下嗜酸粒细胞准确并快速计数的质量控制。方法随机选取75例血液病患者的骨髓涂片标本,要求常规瑞-姬染色骨髓分类嗜酸粒细胞≥3.5%,取材3d内。每份标本中选取两张,分别划入实验组和对照组。实验组标本进行嗜酸粒细胞过氧化物酶快速染色,对照组标本进行常规瑞-姬染色。分别计数嗜酸粒细胞百分数。结果实验组嗜酸粒细胞颗粒染成黑色,对包括中性粒细胞在内的其他细胞染色效果同瑞-姬染色,显微镜下嗜酸粒细胞显示醒目,可快速准确计数。结果与对照组比较,经t检验,P0.05,无统计学差异。结论嗜酸粒细胞过氧化物酶快速染色方法比较常规瑞-姬染色具有快速染色,对嗜酸粒细胞的显示更加醒目的优点,且对包括中性粒细胞在内的其他细胞染色兼有瑞-姬染色的效果。该方法值得推广用于快速骨髓细胞染色,且适用于包括嗜酸粒细胞形态不典型及中性粒细胞颗粒粗大等各种情况下的嗜酸细胞计数的质量控制,从而准确有效地服务于临床诊治。     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

A METHOD FOR SILVER STAINING NERVE FIBRES IN VERY THICK SECTIONS AND IN SUITABLE WHOLE PREPARATIONS

Abstract. In a previous article (1948) the author introduced a rapid method for silver staining nerve fibres in ordinary, mounted paraffin sections (5–25 microns in thickness). By the modification of this method described below, being adjusted to very thick sections (100–300 microns), much more extensive connections of nerve fibres and their ramifications can be demonstrated. The modification can also be used for staining suitable, not sectioned preparations in toto. Some results are shown in photomicrographs.     

Immunocytochemical Staining For the C-Erbb-2 Gene Product In Breast Aspirates: A Preliminary Report

The results of the determination of c-erbB-2 gene expression by immunocytochemical staining of cytological aspirates, prepared by cytocentrifugation, have been compared with paraffin-embedded tissue sections from the same tumour. Our results show equivalent staining in 20/22 cases, with six cases being both scored positive and fourteen cases being both negative. Two samples gave conflicting results. One case was scored as being positive on the cytological aspirate, whereas in the tissue sections taken from the same tumour positive staining was only seen in areas of non-invasive intraduct carcinoma. This sample was scored as being negative. In another case, cytoplasmic staining with less than 50% of the cells showing any positivity was observed in the cytospin sample, with negative staining in the corresponding tissue section. We conclude that expression of c-erbB-2 immunostaining is detectable on cytological preparations prepared by cytocentrifugation but must be interpreted with caution in tumours which may have a large intraduct component or which give predominant cytoplasmic staining.     

Detection of immobilized proteins on nitrocellulose membranes using a biotinylation-dependent system.

Proteins are biotinylated after immobilization on nitrocellulose sheets by reaction with a biotinyl-succinimide ester. The biotinyl residues are visualized by streptavidin-peroxidase-based detection systems either by deposition of a colored formazan dye or by enhanced chemiluminescence (ECL), the latter being 10-fold more sensitive. The sensitivity of the staining procedure is dramatically improved by the inclusion of the reporter deposit technique into the staining procedure: the initially bound peroxidase generates phenolic radicals from biotinyltyramide, enhancing the number of biotinyl residues in the vicinity of the first biotinylation site. Thus the detection limit is lowered to 1 pg of protein with the ECL detection. The new method is compared with silver stain and immunochemical staining in Western blots and furthermore its suitability is demonstrated for 2-D gel electrophoresis.     

Determining subcellular localization of novel drug targets by transient transfection in COS cells

Genomics-based approaches are increasingly being used to identify disease-associated genes that represent potential new drug targets. As a first step in the validation of genes of unknown function, we describe a method for rapidly determining the subcellular localization of the gene product. If an immunotherapeutic approach is being considered, it is of particular interest to identify targets that are either on the cell-surface or secreted. Transient expression in COS cells combined with immunofluorescent staining provides a semi-high throughput method for determining the subcellular localization of multiple targets in parallel. COS cells are ideal for this purpose since: (i) they transfect easily; (ii) the high levels of expression that can be achieved transiently allow detection after 24 h; and (iii) the relatively large size and spread morphology of these cells allows the subcellular organelles to be easily visualized. To evaluate the system, we show prototype staining patterns for known cytoplasmic,secreted, Golgi-associated, endoplasmic reticulum-associated, and plasma membrane proteins, as well as data for novel targets. The localization of novel secretory and cell-surface proteins as determined by immunofluorescent staining, was confirmed by independent methods. This revised version was published online in July 2006 with corrections to the Cover Date.