17β-雌二醇对剑尾鱼卵黄蛋白原的诱导研究

目的研究17β-雌二醇(E2)暴露对雄性剑尾鱼(Xiphophorus helleri)卵黄蛋白原(vitellogenin,Vtg)诱导作用作为环境风险评价(ERA)的有效生物学标记的可行性。方法以E2诱导的剑尾鱼雄性个体的整体匀浆液为材料,采用Sephacryl S-300凝胶过滤层析柱和QSepharose阴离子交换柱从剑尾鱼体内提纯Vtg。结果确定了被纯化的剑尾鱼Vtg在4%~7.5%Native PAGE电泳中相对分子质量为540×103。4%~7.5%Native PAGE电泳后的凝胶分别利用苏丹黑B进行脂蛋白染色、高碘酸-Schiff试剂进行糖蛋白染色和甲基绿进行磷蛋白染色,表明剑尾鱼Vtg是一种富含糖、脂、磷的蛋白。结论表明雄性剑尾鱼卵黄蛋白原的诱导变化可作为环境风险评价(ERA)的有效生物学标记。     

病理组织PAS染色影响因素分析

目的分析病理组织过碘酸雪夫氏(periodic acid Schiff, PAS)染色的影响因素,以期提高其染色效果。方法选取已知含有PAS阳性物质的胃粘膜组织,分别制作成蜡块,通过对比试验,比较高碘酸水溶液不同染色时长、雪夫氏(Schiff)试剂不同配制方法、Schiff试剂不同保存时间对PAS染色质量的影响。结果胃粘膜组织在使用高碘酸水溶液染色11～15min优于其他时长,Schiff试剂热配法优于冷配法,试剂保存2个月内的染色效果优于试剂保存大于2个月者。结论胃粘膜组织使用高碘酸水溶液染色11～15min、热配法配制的Schiff试剂、保存在2个月内的Schiff试剂进行染色效果最佳。     

长白山林蛙输卵管糖蛋白ROGP-Ⅲ的分离纯化及鉴定

通过正交试验确定最佳的粗提工艺,并将林蛙输卵管水提液先后通过盐析,透析,阴离子交换层析、阳离子交换层析和葡聚糖凝胶过滤层析,获得了一种分子量大小约为66 kD的单一蛋白,高碘酸-Schiff试剂法鉴定其为糖蛋白。经HPLC分析,纯度为97.2%,将其命名为ROGP-Ⅲ,经PNGase F糖苷肽酶处理后推测其糖含量约为17%。     

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。     

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。     

PAS染色在骨骼肌糖原贮积症诊断中的应用

目的探讨PAS染色在骨骼肌糖原贮积症诊断中的作用。方法用组织化学方法高碘酸-schiff(periodic acid schiff,PAS)染色方法显示糖原贮积症肌纤维胞浆内糖原的贮积。结果糖原贮积症患者肌纤维胞浆内聚集的红至红紫色颗粒为糖原。结论 PAS染色对于判断细胞内糖原贮积的糖原病和多糖体贮积性疾病的诊断是必要的。     

两种切片方法检测肝糖原染色效果的比较

目的比较石蜡和冰冻两种不同切片用于检测牛蛙肝糖原的效果。方法采用石蜡和冰冻两种切片方法制作牛蛙肝脏切片,高碘酸希夫氏（periodicacid-Schiff＇s,PAS）染色法对肝糖原进行组织化学染色,光密度分析糖原含量。结果PAS染色显示肝糖原为紫红色或红色颗粒,冰冻切片中糖原颗粒明显大于石蜡切片,光密度分析显示,石蜡切片中糖原流失明显,与冰冻切片相比,糖原流失了约28％。结论两种切片均可用于糖原检测,冰冻切片制作环节较少、耗时短且染色过程中糖原不易流失。     

聚丙烯酰胺凝胶电泳脂蛋白带的耐尔氏蓝染色法

目前聚丙烯酰胺凝胶电泳用考马斯亮蓝G或R染蛋白带、用过碘酸-Schiff试剂染糖蛋白带,均取得了满意的结果.至于用油红(OR)、苏丹黑(SB)对琼脂、醋酸纤维膜等凝胶中的脂蛋白带的染色鉴定是成功的,但对聚丙烯酰胺凝胶中的脂蛋白带的染色则不够满意.我们应用耐尔氏蓝[Nile’s blue] 染色法染盘状聚丙烯酰胺凝胶电泳的脂蛋白带取得了较好的结果.现介绍如下:     

HE染色细胞核灰染的处理方法比较研究

目的探讨HE染色细胞核灰染的处理方法,提高HE染色质量。方法收集10例HE染色发灰的胃粘膜、肠息肉和子宫内膜等不同类型的组织,重新切片,通过调整苏木素染色条件以及染色前对组织进行修复处理,设对照组,比较各种处理方法的染色效果。结果通过延长时间、水浴加热和微波加热等调整苏木素染色条件的方法以及水煮修复处理法能够不同程度地促进核染色,但灰染现象未能彻底解决。PBS、EDTA、柠檬酸分别采用高压和微波修复处理均能较好地改善HE染色细胞核灰染现象,以PBS微波处理法效果最佳。结论 PBS微波处理法是HE染色细胞核灰染现象的一种有效处理方法。     

以偶氮偶联(azo-conpling)及粘蛋白染色的对比作脑垂体前叶诸型细胞的组织化学鉴别

根据 Pearse 氏偶联四氮盐反应(coupled tetrazo-nium)染脑下垂体,其嗜酸嗜硷两种细胞皆可染色,仅前者着色较深,故不宜于组织学鉴别之用。作者鉴于嗜硷细胞的向性腺及向甲状腺激素皆系粘蛋白(mucopro-tein),故染粘蛋白的高碘酸白复红法(periodic acid-leukofuchsin)可作力重氮蓝B(diazo llue B)或重氮蓝B和H酸(H acid)联用的对此染色。作者也鉴于用重氮蓝B可加强嗜酸性细胞对于某些染料的亲和力(如固绿FCF)而描述了应用固绿 FCF(fastgreen FCF)的偶氮偶联染色法。     

Bassam和Sanguinetti银染方法在SRAP和TRAP标记中的比较研究

银染作为一种重要的DNA染色方法,对分子标记检测有重要影响.SRAP标记和TRAP标记是两种较为新型的分子标记,近年来得到了广泛应用,尤其在缺少遗传图谱的物种中应用价值更大.以普通烟草种(Nicotiana tabacum L.)烤烟和香料烟品种作为供试材料,对Bassam和Sanguinetti两种银染方法,在标记检测效率、成本及染色效果等方面进行了研究,并对其在SRAP标记和TRAP标记中的应用进行了探讨.结果表明,Sanguinetti银染法比Bassam银染更为简便、经济,染色照片的色阶图说明Sanguinetti染色方法的背景与扩增带区分明显,能够清楚地读带;且该方法能够扩增出很好的SRAP和TRAP标记谱带.因此,推荐在SRAP和TRAP标记检测中采用Sanguinetti银染方法.     

About the mechanism of interference of silver staining with peptide mass spectrometry

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Onset of nucleolus organizer activity in early mouse embryogenesis and evidence for its regulation

Ovulated mouse oocytes and preimplantation embryos were examined for NOR activity by means of selective silver staining. Evidence of the first staining activity appeared in two cell embryos, which was later followed by an increase in nucleolar activity, whereas the ovulated oocytes and pronuclei showed no such activity whatsoever. The staining of chromosomes was restricted to the nucleolus organizing region. Our results agree with earlier observations that genes for ribosomal RNA (rRNA) are transcribed as early as in the 2-cell stage in mouse embryogenesis. In addition to the nuclear staining we also observed some silver staining within the cytoplasm, at least from 4-cell stages onwards. Cytoplasmic staining was resistant to incubation with cycloheximide and actinomycin D. Nuclear staining was depressed, or even totally blocked, after actinomycin D incubation but was not blocked by cycloheximide. The onset of silver staining depends not on a specific embryonic stage but on the time interval following ovulation. This appears to indicate that the initiation of ribosomal cistrons is regulated by molecules which are activated or synthesized within the oocyte soon after ovulation.     

Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility

A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1 h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1 h with a detection limit of 0.2 ng/band.     

Experimental studies of mechanisms involved in methods demonstrating axonal and terminal degeneration

Factors influencing the consistency and specificity of the staining of neuronal degeneration products were studied in brain sections by varying systematically the composition of solutions used in the steps which are common to the degeneration methods. The formation of nuclei of metallic silver was determined either by physical development of 110Ag, after dissolving reducible silver by acetic acid. In degenerating axons metallic silver nucleic are formed by their own reducing groups in the first (acid) and in the second (alkaline) impregnating bath. The first impregnation turned out to be sufficient to produce complete staining of degenerating axons. The reducing capacity of normal axons and myelin can be suppressed by oxidation or by lowering the pH of the impregnating solution. Degenerating axon terminals are not able to reduce silver ions in either of the impregnating baths. Rather, the metallic silver nuclei initiating their staining are formed in the Nauta reducer by interaction of its reducing agent (formol) with silver ions which had been trapped in the tissue during the impregnation. Thus the nuclei are enlarged to microscopic visibility by a nonstandardized physical developer coming about from the Nauta reducer and the silver ions transferred with the sections. In this reaction catalytic sites in degenerating terminals as well as ammonium ions and the alkali reserve of the tissue play an important role. On the basis of the present results it was possible to stabilize the conditions for staining degenerating axons and degenerating axons terminals in two separate staining procedures detailed in following papers.     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.     

Histochemical investigations on the nature of large blood vessel endothelial and medial argyrophilic lines and on the mechanism of silver staining

Summary A study of the mechanisms involved in silver staining of blood vessels has been performed on the rabbit and rat aorta and vena cava, both in fixed and unfixed states. Pretreatment with cationic detergents, organic solvents, and solutions containing free iodide ions inhibited the silver staining. Anionic or neutral detergents, oxidizing agents, binders of such ions as Ca⁺⁺, Mg⁺⁺ and SO ₄ ^- failed to inhibit the staining. Staining of the intercellular gaps between endothelial cells and between smooth muscle cells could also be obtained if vessels were treated with a cationic detergent and bromocresol green, or by a modified Hale's colloidal iron technique. Silver lines could be returned to dechlorinated vessels, if treated with sodium chloride before silver nitrate staining, but not vice versa; by an extended treatment with dilute silver nitrate or with gold chloride following normal silver nitrate staining; and by treatment with heparin prior to silver staining. Dark chamber experiments have demonstrated that a photographic developer can take the place of light in the silver staining procedure and that a photographic fixer has the same effect on vessel silver staining as dechlorination.The obtained results have led to the hypothesis that silver staining of vessels occurs in two stages. In the first silver ions from silver nitrate are bound by polyanions located primarily in the intercellular gaps, and then reduced. This produces a network of reduced silver grains which, however, are still too sparsely aggregated to be visualized. Chloride ions in the tissues also bind and precipitate silver ions preventing their removal in subsequent rinsing procedures. In the second stage light (or a photographic developer) reduces the silver ions in silver chloride, producing a visible accumulation of metallic silver, but only around the silver grains reduced during the first stage, analogous to the photographic process.The possible existence and function of an intercellular cement substance is discussed in light of the evidence for the presence of polyanionic groups in the intercellular gaps.     

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes

Summary A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.