多种特殊染色法在骨关节炎组织形态学研究中的应用比较

目的探讨多种特殊染色法在骨关节组织中的染色规律及其在骨关节炎形态学研究中的应用价值。方法 6月龄健康新西兰大白兔20只,随机分为正常组和造模组各10只,根据改良Hulth法造模,6周后膝关节取材。对标本固定、脱钙后进行石蜡包埋和切片。分别采用HE、番红-固绿、AB-PAS、甲苯胺蓝、Van Gieson染色和Mallory染色,观察骨关节组织的形态学变化,并对几种染色方法进行比较。结果 HE染色显示关节一般组织形态结构,可见模型组关节软骨和软骨下骨发生骨性关节炎病理变化;番红-固绿染色法中软骨和软骨下骨的界限（黏合线）以及潮线显示清晰,软骨基质中糖胺聚糖含量减少,纤维成分增多;AB-PAS染色显示骨关节炎软骨基质糖胺聚糖尤其是酸性糖胺聚糖含量减少;甲苯胺蓝染色显示骨关节炎软骨的酸性糖胺聚糖减少;Van Gieson染色和Mallory染色可显示骨关节组织中的胶原纤维,但组织结构界限不够清晰。结论在骨性关节炎的组织形态学研究中,通过常规HE染色,结合番红-固绿染色法和AB-PAS染色法,能较客观全面地获得关节组织形态学相关信息。     

改良革兰染色法在显示石蜡切片真菌中的应用

目的建立真菌的病理切片改良革兰染色法。方法选取确诊真菌感染的组织蜡块,切若干空白片,通过苏木素-伊红染色（HE）、高碘酸-无色品红染色（PAS）、六胺银染色（GMS）、改良革兰染色后,比较改良革兰染色法的染色效果。结果改良革兰染色法的染色效果好,该法具有易操作、染色效果稳定等优点。结论改良革兰染色法能够清晰地显示出组织切片中的真菌,并且染色效果稳定,在检测组织中的真菌时可发挥重要的辅助诊断价值,具有广泛的应用前景。     

移植肾活检组织C4d免疫组化方法的比较

目的探讨移植肾活检组织C4d免疫组化检测方法 ,寻求其实验的标准化。方法 121例移植肾活检和10例供肾标本根据ALPCO抗体手工免疫组化结果将病例分为四个组:C4d弥漫阳性组、局灶阳性组、阴性组和供肾组,采用ALPCO和ABCAM两种C4d抗体,石蜡切片行手工免疫组化和全自动免疫组化机器套染技术(以下简称机器套染),比较两种抗体和染色方法的染色结果。结果 ALPCO和ABCAM两种抗体手工免疫组化和机器套染在肾小球毛细血管基底膜(GBM)和肾小管周围毛细血管(PTC)均有良好着色,在20例ALPCO抗体手工免疫组化为弥漫阳性组标本中,染色强度在+至+++之间,而机器套染均为+,有7例标本机器套染染色强度低于手工染色,但并不改变染色的弥漫程度,另有3例其机器套染为局灶阳性;而ABCAM抗体其手工免疫组化和机器套染染色强度除1例为阴性外,其余均为+,有3例手工免疫组化和4例机器套染为局灶阳性。对于ALPCO抗体手工免疫组化为局灶阳性组的标本,其机器套染和AB-CAM抗体染色重复性较差:其机器套染有1例为阴性,而ABCAM抗体手工免疫组化有2例、机器套染有3例为阴性。ALPCO抗体手工免疫组化阴性组和供肾组标本,其机器套染和ABCAM抗体染色均为阴性,重复性良好。同时机器套染与手工免疫组化相比,拥有更好的染色背景,而且大大缩短了染色时间,更有利于急诊肾活检和免疫组化实验的标准化。结论虽然ALPCO和ABCAM两种抗体染色效果有小的差异,但总体重复性良好。ABCAM抗体在染色过程中尤其是机器套染应注意防止假阴性。手工免疫组化和机器套染联合使用为C4d免疫组化染色提供更为快捷、可靠的技术保障。     

蛋白负染方法在蛋白质组学中的应用评价

为评价蛋白质负染方法在蛋白质组学分析中的应用,采用负染和考马斯亮蓝染色两种方法对同一样品的双向电泳胶进行染色,取相对应的8对蛋白点,并进行胶内酶解及MALDI-TOF/TOF分析,比较两种方法与质谱的兼容性。图像分析显示,负染方法展示出的蛋白点更多,但三维峰图不如考染明晰;质谱结果显示,8个负染蛋白点中有7个鉴定结果有效,8个考染蛋白点鉴定结果均有效。因此可以得出以下结论:负染的灵敏度高于考染,与质谱的兼容性良好,适用于建立双向电泳参考图谱的研究;但负染后的胶图不适于进行蛋白点丰度对比分析。     

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,最近发现了延长赖失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。     

Two-color immunostaining of liver fine needle aspiration biopsies with CD34 and carcinoembryonic antigen. Potential utilization in the diagnosis of primary hepatocellular carcinoma vs. metastatic tumor

OBJECTIVE: To examine immunohistochemical staining of cell block material with antibodies against vascular marker CD34 and polyclonal carcinoembryonic antigen (pCEA) for their clinical utility as part of a 2-color staining protocol in fine needle aspiration (FNA) biopsy of liver masses to distinguish metastases from primary hepatocellular carcinoma (HCC). STUDY DESIGN: The authors obtained cell block material from 96 liver FNAs and performed simultaneous (i.e., "dual-color") immunohistochemical staining utilizing antibodies against vascular marker CD34 and pCEA. Cases were blinded and evaluated by the authors for staining pattern and intensity. A consensus was obtained, the results were unblinded, and the diagnoses were correlated. RESULTS: After staining, 89 cases had sufficient tissue for evaluation. Of the 19 HCC cases, 16 (84%) showed peripheral staining with CD34, and 13 (68%) showed a canalicular or mixed canalicular-cytoplasmic staining pattern for pCEA. Thirteen cases (68%) showed staining for both antigens. All HCC exhibited immunostaining for at least 1 antibody in an appropriate staining pattern. Of the 67 cases of metastatic malignancy, 5 (7%) showed a predominantly transgressing pattern of CD34 staining, 43 (64%) showed a predominantly cytoplasmic or mixed cytoplasmic-canalicular pattern of pCEA staining, and 2 cases (3%) showed staining for both antigens in a transgressing CD34 pattern and cytoplasmic pCEA pattern. None of the 3 normal liver tissue blocks showed staining with either antigen. CONCLUSION: Two-color immunohistochemical staining of liver cell block material obtained by FNA with antibodies to CD34 and pCEA can be helpful in differentiating metastatic tumors vs. primary HCC.     

植物细胞壁组织化学定位染色方法和技术的比较研究

利用光学和荧光显微镜比较研究几种植物细胞壁组织化学定位染色方法和技术,结果表明：（1）硫酸消化法和硫酸氢黄连素-苯胺兰对染法研究凯氏带,对取材时间和部位要求高,建议两种方法配合使用,可相互印证是否具凯氏带;（2）苏丹7B染色法,蓝色激发光下不染色和硫酸氢黄连素-苯胺兰对染研究细胞壁栓质层3种方法中,不染色蓝色激发光下结果比苏丹7B染色法敏感显色,但苏丹7B染色法在普通光学显微镜下观察较为便捷;（3）木质化细胞壁染色方法中硫酸氢黄连素-苯胺兰对染法比间苯三酚-盐酸染色法易显色观察;（4）甲苯胺兰快速染色细胞壁取代常规苏丹Ⅲ/Ⅳ法,细胞边界和层次更清楚。     

A simple staining method for cryptosporidian oocysts and sporozoites

A useful staining method for detection of cryptosporidian oocysts and sporozoites was developed and described. The modified Kohn's one-step staining technique with an additional modification, i.e., longer staining time and higher staining temperature than those originally described, was tested on fecal smears from cats infected with Cryptosporidium sp. By this improved staining procedure, the oocyst appeared as a slightly oval body containing four internal sporozoites colored blue to blue-gray. The oocyst wall was stained dark green to black. The morphological feature of the oocyst was recognized much more clearly by this staining technique than by such currently used techniques as acid-fast and Giemsa staining. This staining procedure proved to be simple and less costly and to secure a good preservation.     

检测聚丙烯酰胺凝胶中蛋白质的3种染色方法比较

用考马斯亮蓝染色、银染色、铜染色等 3种方法对同一种蛋白质染色的灵敏度、快速性进行比较 ,得出 3种蛋白质染色方法的优缺点 ,为蛋白质电泳染色合理选用不同方法提供依据     

Enzymatic Elimination of Collagen in the Alcian Blue Staining of Connective Tissue Ground Substance

Collagenase pretreatment of frozen-dried sections permits Alcian blue staining of mucopolysaccharides of connective tissue ground substance without the interference of collagen staining. Hyaluronidase elimination of Alcian blue staining confirms mucopolysaccharide as a substrate of the staining reaction.     

介绍一种用卡介苗进行改良抗酸染色的实验教学方法

利用卡介苗取代结核分枝杆菌阳性痰液作为实验标本,使用改良后的抗酸染液进行抗酸染色实验教学。卡介苗水溶液为标本,石炭酸复红染色液中加入5%的Tween-80进行抗酸染色。染色后,卡介苗标本片与结核菌阳性痰液标本比较,菌体形态与染色均无明显差异。此法具有染色效果好、标本来源方便、安全无污染等优点,满足抗酸染色实验教学需要。     

Immunolocalization of zona pellucida antigens in the ovarian follicle of dogs, cats, horses and elephants

A comparative evaluation of the location of immunoreactive porcine zona pellucida (pZP) glycoproteins was performed with polyclonal rabbit anti-pZP antibodies on ovarian sections of the dog, cat, horse, and elephant. For this, formalin (light microscopy) and glutaraldehyde (transmission electron microscopy [TEM]) fixed ovarian sections were incubated with antibodies raised against highly purified pZP. Staining patterns were determined with diaminobenzidine (DAB) at the light level. The dog ZP had a distinct staining distribution that is characterized by intense staining around the periphery of the ZP and the oolemma and less dense staining throughout the width of the ZP. In dog follicles that contained multiple oocytes, there were oocytes of identical and dissimilar stages. Cat ovarian sections showed uniform staining of the ZP. Horse results showed uniform staining of ZP and ooplasm, and granulosa cells (GC). Elephant sections showed staining of the ZP with dense staining at the oolemma, as well as staining of the ooplasm. In all species the staining of the ZP was not evident until GC differentiation. In all cases there was no staining of ovarian tissue with control normal rabbit serum. Specific staining patterns of ZP were evaluated by TEM and immunogold staining. The immunogold-linked anti-pZP antibodies stained the ZP matrix in all species. There was staining of ooplasm organelles suggesting that ZP secretion originates from the oocyte of the dog and cat. In addition, follicular and ZP measurements were taken that allowed accurate characterization of follicle stage. These findings suggest that in all four species the ZP is recognized by anti-pZP antibodies and there is also evidence to suggest the possible origins of ZP glycoproteins.     

Demonstration of myoglobin and CK-M in myocardium. Comparison of five fixation methods and three immunohistochemical techniques

The results of immunohistochemical staining vary depending on the tissue, fixative, antigen-antibody system, and immunohistochemical staining methods used. The purpose of this study was to evaluate the effect of different methods of fixation, different antigen-antibody systems, and different immunohistochemical methods on immunohistochemical staining of myocardium. Samples of normal fresh canine myocardium from six dogs were fresh frozen and fixed in 10% neutral buffered formalin, Bouin's, Bayley's and Carnoy's fixatives. Immunohistochemical staining for myoglobin and creatine kinase M was performed using the ABC (avidin-biotin complex) and indirect peroxidase-antiperoxidase (PAP) techniques. Tissues fixed in formalin showed the most intense specific staining for both antigens with the least background and nonspecific staining. All other fixation methods and frozen section techniques gave a more variable degree of specific positive staining and substantial background staining and/or nonspecific staining. ABC and PAP techniques gave similar results with both antigen-antibody systems and with each fixation method. Thus, no differences in specificity or sensitivity were observed between ABC and PAP techniques. Differences in staining intensity and pattern were related primarily to differences in fixation methods.     

皮肤组织神经髓鞘几种染色方法的比较

对皮肤组织神经髓鞘应用髓磷脂硷性蛋白myelin basic protein简称MBP）免疫组织化学,劳克坚牢蓝(Luxol fast blue,简称LFB）,砂罗铬花青（solochrome cyanine简称SC）,KOH-HIO4-Schiff四种不同的染色方法进行染色比较,结果显示,MBP法具有特异性强,背景干净,对比清晰等特点。SC、Schiff法髓鞘着色效果亦较好,但背景中其他组织也着色,只要分化适当,LFB法髓鞘着色鲜明,且有一定特异性。     

Stain-Free total protein staining is a superior loading control to β-actin for Western blots

Semi-quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, Ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain-Free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain-Free staining as an alternative to β-actin or the protein stain Ponceau S showed that Stain-Free staining was superior to β-actin and as good as or better than Ponceau S staining as a loading control for Western blots.     

A protocol for combining proliferation, tetramer staining and intracellular cytokine detection for the flow-cytometric analysis of antigen specific T-cells

Flow-cytometry can be used in different ways in order to analyze or enumerate antigen specific T-cells. The three basic principles are direct staining of the T-cell receptor using so called tetramer reagents, staining intracellular cytokines following antigen-specific ex vivo T-cell activation or staining with dyes that are incorporated (increase in staining) or distributed between daughter cells (decrease in staining) upon proliferation in response to a specific antigen challenge. Each system has its advantages and disadvantages. Here we demonstrate that tetramer staining, cytokine flow cytometry and staining with CFDA-SE can be combined permitting the analysis of proliferation and cytokine production with a subset of T-cells specific for a single peptide antigen.     

Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.

Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.     

苏木精染色液的改进和使用

报道了一种新的特制苏木精染色液的配制、使用方法、染色效果及其使用价值。实验表明,该染色液全面而明显地优于通用的Harris染色液。     

Victoria blue B--a nuclear stain for cytology. A cytophotometric study

The aim of this study was to investigate the staining characteristics of Victoria Blue B in alcohol solutions. Cytological specimens (liver and spleen tissue imprints, blood smears) were stained with methanol solutions of commercially available Victoria Blue B-Cl and with pure Victoria Blue B-BF4. The dye concentration, staining time, and protone concentration of the dye solution were varied. The dye solutions were characterized using spectrophotometry and thin-layer chromatography. Cytophotometry and image analysis were used to quantitate the staining pattern of cell nuclei. Feulgen-stained slides were used as controls. Victoria Blue B-BF4 gave excellent nuclear staining exhibiting a quantitative dye-substrate relationship, whereas commercial dyes resulted in lower staining intensity and less distinct nuclear texture. Dye concentration and staining time were, over wide ranges, not of critical importance for the quality of the staining. Under certain staining conditions, only cell nuclei were stained, with the background remaining completely unstained. We presume that, in alcohol solutions, Victoria Blue dye binds as a neutral dye molecule in conjunction with its anion. Victoria Blue B-BF4 staining provides a simple and reproducible staining technique for cytology which is suitable for use in automated cell-pattern recognition.     

莱茵衣藻和斜生栅藻蛋白核的染色方法优化

蛋白核（pyrenoid）作为真核藻类重要的、相对独立的固碳结构,在CO2高效浓缩和固定过程中发挥中重要作用,可为开发高效生物固碳技术提供理论支持。染色法是快速观察蛋白核的最佳方法之一,有助于深入研究蛋白核的结构和功能,但目前对染色法仍缺乏系统研究。本文以较易染色的莱茵衣藻（Chlamydomonas reinhardtii）和较难染色的斜生栅藻（Scenedesmus obliquus）为材料,探讨了碘染和溴酚蓝（BPB）染色观察蛋白核的方法。结果表明,碘染法最适用于蛋白核外周淀粉观察,最适染色液浓度和时间分别是0．1％～0．2％（w／v）和5min。热激预处理和溴酚蓝染色相结合的蛋白核染色效果更好,优化条件为：70℃水浴热激40s,0．05％（w／v）BPB染色15min。本方法为研究微藻蛋白核提供了一种快速有效的直接观察方法。