Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence

The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.     

Autofluorescence removal using a customized filter set

Quantitative fluorescence microscopy is severely hindered by intrinsic autofluorescence (AF). Endogenous fluorescent molecules in tissue and cell samples emit fluorescence that often dominates signals from specific dyes. This makes AF removal critical to the development and practice of quantitative fluorescence microscopy. In this study, we showed that AF signal could be separated from specific signal using a customized filter set. The filter set used the same excitation and beam splitter as the standard filter set, but the emission filter was red‐shifted 40–60 nm from the peak of the specific dye. This filter set configuration collected mostly AF with minimum contribution from the specific dye. A linear transformation of AF images was required to correct for the difference in exposure and filter configuration. The constants (slope and intercept) in linear transformation were obtained through a pixel to pixel comparison between AF images (no staining) obtained by the standard filter set and the customized AF filter set. After staining of specific dye, the standard filter collecting target dye spectra was used to capture both target signal and AF, whereas customized filter was used to capture only AF. AF removal was accomplished by subtracting the linear transformed AF image from the image obtained from the standard filter. To validate our approach, we examined weak staining of androgen receptor in an AF abundant prostate tissue sample. Our method revealed a similar but cleaner nuclear staining of androgen receptor in a specimen, when compared to a traditional autofluorescence removal method. Microsc. Res. Tech., 76:1007–1015, 2013. © 2013 Wiley Periodicals, Inc.     

奶牛乳腺发育过程中整联蛋白α6、β4表达定位的研究

为了解奶牛乳腺发育与整联蛋白α6、β4表达的规律,作者取荷斯坦奶牛乳腺组织,制备冰冻切片,进行免疫荧光标记,在激光共聚焦显微镜下观察。结果显示,整联蛋白α6的荧光信号存在于乳腺导管和腺泡基底一侧细胞的基底细胞质膜上,整联蛋白β4主要分布于腔上皮细胞的基底侧、相邻腔细胞间侧膜区域以及围绕在腔上皮细胞外周的肌上皮细胞膜上。整联蛋白α6、β4在肌上皮细胞和基底细胞层中也可被检测到,但是细胞腔内几乎不能被检测到。从整联蛋白α6、β4的共定位的部位推测,α6β4异二聚体在导管和腺泡基底侧分布,整联蛋白α6在面向导管和腔上皮细胞中表达,但不完全以α6β4异二聚体形式存在。     

超导体包被免疫荧光试纸法同时测定玉米中脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮适用性研究

为了能同时快速测定玉米中脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和玉米赤霉烯酮(Zearalenone,ZEN)两种毒素,采用基于超导体包被的免疫荧光快速定量检测技术,检测了多个玉米样品,结合加标回收率、稳定性、检出限、精密度等指标,并与液相色谱法的检测结果进行比较分析,评估方法的适用性。结果表明,超导体包被的免疫荧光试纸法检测DON含量在100～2 500μg/kg,检测ZEN含量在5～200μg/kg的范围内线性良好。DON在500～2 000μg/kg添加水平范围内,回收率为103.72%～108.17%,ZEN在50～150μg/kg添加水平范围内,回收率为97.81%～111.27%。该方法于液相色谱法检测结果对比,DON相对偏差在3.72%～8.17%,ZEN相对偏差在2.19%～11.27%,均低于液相色谱法允许偏差23%和15%,超导体包被的免疫荧光试纸法是一种有效、实用、快速、定量的分析方法,能满足同时检测玉米中DON和ZEN的要求。     

Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls

This article describes a practical technique for permeabilization of higher plant cell walls, which is usually one of the first steps required for immunolocalization of cellular components (and other cytological methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It replaces the use of wall degrading enzymes or the need to employ laborious sectioning or other mechanical means for providing access of probes to cells. Freeze-shattering retains the integrity of whole tissues and cells surprisingly well and thus is especially useful when used in conjunction with confocal laser scanning microscopy for recording the three-dimensional arrangement of cytoskeletal elements in relation to cell shape. In this article, we demonstrate the effectiveness of this technique for anti-tubulin and anti-actin immunofluorescence and for rhodamine phalloidin labelling of the cytoskeleton in various higher plant tissues including onion root tip and bulb scale epidermis, Tradescantia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells.     

密度梯度分离纯化/免疫荧光技术检测饮用水中“两虫”

采用膜溶解、密度梯度分离纯化结合免疫荧光染色法对饮用水中的“两虫”（隐孢子虫和贾第鞭毛虫）进行定性与定量检测，以大幅降低检测成本，简化操作。结果表明，使用Percoll-蔗糖介质可取得良好的分离纯化效果，其卵囊和孢囊初始回收率分别为56％和35％，远高于EPA1623方法的要求。利用该方法对河网型水源水及水厂滤后水进行了检测，发现10L水源水中有1～3个隐孢子虫和0～2个贾第鞭毛虫，但在50L滤后水中均未检出“两虫”。     

THE IMMUNOFLUORESCENT STAINING TECHNIQUE FOR THE DETECTION OF WILD YEASTS - PRACTICAL PROBLEMS

An apparent heavy wild yeast infection in pitching yeast has been detected using the immunofluorescent detection method. This infection could not be detected by conventional liquid forcing or plating techniques. The yeasts responsible were isolated and identified as Saccharomyces cerevisiae. The yeasts were very similar to the pitching yeast but varied in a number of respects associated with the cell wall such as flocculation character and giant colony morphology. The results suggest that the immunofluorescent-positive (IP) yeasts are variants of the culture yeast. As a result of this work it is felt that although immunofluorescence is of value for the rapid detection of infection, it must always be used in association with more conventional microbiological techniques.     

Rapid identification and characterization of peroxisomal assembly mutants in Yarrowia lipolytica

We describe the isolation and characterization of p eroxisomal a ssembly mutants in the genetically manipulable yeast Y arrowia lipolytica (pay mutants). These mutants were initially identified as oleic acid-non-utilizers by their inability to grow on oleic acid, the utilization of which requires peroxisomal β-oxidation enzymes. Identification of a subset of oleic acid-non-utilizers as pay mutants was obtained by a rapid immunofluorescence procedure using antibodies to the peroxisomal targeting signal Ser-Lys-Leu-CO₂H. Punctate structures characteristic of peroxisomes were not detected in pay mutants using this technique. This rapid identification by immunofluorescence should be generally applicable to the selection of peroxisomal assembly mutants in other yeasts. To take advantage of the pay mutant system, we constructed a genomic library in the autonomously replicating vector pINA445 and developed an efficient and rapid electroporation procedure for the functional complementation of these mutants. We have been successful in functionally complementing two independent pay mutants. Molecular analysis of these and other complementing genes will allow for characterization of some of the cellular elements involved in peroxisomal assembly.     

Immunolocalization of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution fixation and methacrylate de-embedment

Immunolocalizations in whole mounts of walled cells require an empirically derived, species-specific permeabilization strategy. Particularly when cell walls are recalcitrant to enzymatic digestion, as with many fungi, permeabilization can degrade structure and reduce or destroy antigenicity. To avoid these potential problems, hyphae were freeze-substituted, embedded in a methacrylate resin, thick-sectioned and de-embedded prior to immunolocalization. The cytoskeletal proteins actin and β-tubulin were labelled via indirect immunofluorescence in hyphal tip cells of Magnaporthe grisea , and Trichoderma viride . Well-defined punctate actin plaques were observed concentrated at the subapical cell periphery in both species, especially near the hypha–substratum interface. In addition, a Spitzenkörper core and less intense cytoplasmic binding were observed. Immunoelectron microscopy confirmed localization of actin in the Spitzenkörper core and filasomes. β-Tubulin was immunolocalized as both linear arrays and punctate dots via epifluorescence microscopy. Analysis of 450–550-nm-thick serial sections contributed to improved resolution by the reduction of out-of-focus blur. Overall, the freeze substitution methacrylate de-embedment technique provided superior preservation, serial section analysis, and improved antibody penetration and resolution of these cytoskeletal proteins. The technique will be a valuable tool for use with a variety of reporter molecules in cells of fungi.     

应用生物素—亲和素间接免疫荧光法检测支原体

应用生物素-亲和素系统导入间接免疫荧光法,对不同株的支原体进行了初步检测。结果生物素-亲和素间接免疫荧光法(ABS—IFA)比普通间接免疫荧光法敏感度高百倍以上。在ABS—IFA方法中,阳性支原体部位苹果绿染色明亮、清晰,而背景浅且较干净。