一种新型细胞微阵列的制作和应用

为了高通量地检测大量培养细胞中基因原位表达, 发明了一种制作细胞微阵列的新方法,成功地制作含20种细胞系共100个供体细胞石蜡混合物点阵的细胞微阵列.免疫组化检测P53, P21, PTEN、P16基因在细胞微阵列中的蛋白质表达.原位杂交检测BRD7、NGX6 基因在细胞微阵列中mRNA原位表达.建立了P53、P21、PTEN、P16蛋白和BRD7、NGX6 mRNA 在不同培养细胞中的原位表达谱.细胞微阵列为基因功能研究提供一种新的高通量工具.细胞微阵列可广泛用于DNA、RNA和蛋白质水平上的基因原位表达研究.细胞微阵列还可用于筛选药物作用靶标的研究.     

常规染色与特殊染色相结合的多种肌组织混合切片的制作

目的探索一种结合常规染色与特殊染色的多种肌组织混合切片的制作方法。方法取实验动物狗的平滑肌、心肌、骨骼肌。所有组织均采取单一包埋制作蜡块。另取心肌组织块,制作显示心肌闰盘的特殊块染标本的蜡块。将四种蜡块切片的蜡带进行组合粘片,同时粘在一张载玻片上。对除心肌闰盘外的三种肌组织切片进行苏木素伊红染色。再将所有切片浸入二甲苯,最后共同封片。结果获得常规染色与特殊染色相结合的多种肌组织混合切片。可在一张混合切片上分别观察到平滑肌、心肌、骨骼肌、心肌闰盘四种结构。不同标本的形态结构保存完好,对比非常清晰。结论采取单一包埋与不同蜡带的组合粘片法,过程简单,操作容易,适合制作常规染色与特殊染色相结合的多种肌组织混合切片。     

组织微阵列的原理及应用

组织微阵列是将若干石蜡包埋块上的各种典型组织或细胞转移到一个新石蜡块上重新构建微型化高通量组织阵列的方法,可以有效地进行各类临床病理组织的原位观察和研究。组织微阵列技术可以在一块薄片上同时排列几百个样品,进行高通量的基因和蛋白质分析,且能够充分利用组织资源,将多个样品的平行实验一次完成。在分析RNA和某些蛋白质时,石蜡包埋组织的使用受到限制。为了解决这个问题,发展了冷冻微阵列的方法。该方法快速便捷,效率很高,为使用临床标本及其相关的病理数据库来进行基因和蛋白质水平的研究提供了机会。该阐述了来自于组织和细胞系的微阵列的制备方法和技术特点,并概述它们在临床研究中的应用以及发展前景。     

人胎肝组织制作蜡块脱水时间的探讨

目的:探讨人胎肝组织制作蜡块的适宜脱水时间.方法:应用16印象例不同发育阶段的人胎肝组织标本,通过HE石蜡制作技术进行探索性研究肝组织适宜的脱水时间.结果:人胎肝组织蜡块内组织浸蜡充分,与周围石蜡紧密相连.经HE染色后,在光镜下可见肝组织结构清晰,肝细胞形态完整.结论:人胎肝组织适宜的脱水时间是优质蜡块制作的关键环节.     

手工制作组织芯片方法的改良及其在肝细胞癌研究中的应用

组织芯片（tissue chip）,又称组织微阵列（Tissue microarray,TMA）,是指通过构建组织阵列蜡块将数十至数千个小组织排列在一张载玻片上而制成的组织阵列切片。作为生物芯片的新成员,具有体积小、信息量大、简便快捷的特点,并能将分子生物学和组织形态学相结合,满足基础和临床研究工作者的需要,具有广泛的应用前景。然而自动组织芯片制备仪价格昂贵,尚不能普及。[第一段]     

怎样制作好实验动物病理的石蜡切片

有关动物实验病理组织切片的制作文献报告甚少,本文小结了我们十多年来制作不同种类的实验动物病理组织切片的经验,对必须选用缓冲中性甲醛固定液和控制各类组织的脱水、透明浸蜡时间以及动物组织切片的过程特殊处理事宜作了说明。     

植物石蜡切片包埋、修蜡过程的改进

传统的石蜡切片包埋、修蜡操作过程耗时费力。采用金属框替代纸质包埋盒进行组织包埋,将蜡块冷水冷却改为自然冷却,趁蜡块未完全变硬之前进行快速修整。该方法不仅提高了实验效率,而且修出的蜡块工整一致,同时对后面的切片过程无任何影响。     

病理石蜡制片中试剂标准化操作的应用体会

近年来,随着经济的发展,蜡块制作由人工操作转向全自动密闭式脱水机的广泛使用,组织的固定、脱水、透明、浸蜡已进入恒温、恒湿化操作,有效地提高和稳定了病理制片的质量,但在各实验室石蜡HE制片中的试剂使用尚未统一实现标准化操作,影响了制作的蜡块质量稳定性。现将石蜡切片试剂标准化使用问题作如下探讨：     

自制组织芯片穿刺工具及芯片手工制作技巧

组织芯片（tissue chip TC）技术是将数十个乃至上千个的组织样本,整齐有序地排列在同一张载玻片上,而形成的微缩组织切片;又称组织微阵列（tissue microarray TMA）技术。组织芯片的制作方式主要有石蜡切片法和冷冻切片法,目前以石蜡切片法为常用。现在还有商品化的组织芯片制作仪,如美国（Tissue Arrayer,Beecher Instrument,USA）生产的,但这套产品价格昂贵。所以,手工制作技术更经济、实用,其方法的改进层出不穷。我们采用一次性穿刺针手工制做穿刺工具,用普通石蜡包埋样本,制做组织芯片,收到了满意的效果,现将制做主要程序和技巧介绍如下。     

半月板切片制作方法的改进

半月板主要由纤维软骨构成 ,具有一定的硬度 ,经二甲苯透明和高温石蜡浸蜡后更坚硬易碎 ,不易切出完整的组织切片。国内外虽有此项方法学研究的报导 ,但较复杂 ,我们就此在常规切片制作方法的基础上进行了改进研究。我们经过 4 0例试验 ,摸索出一种简易有效的半月板切片制作方法。1　材料及固定1.1　材料　选用成年大白兔的半月板。1.2　固定　组织块用 10 %中性甲醛或波氏液固定 3天以上。2　组织蜡块的制作2 .1　脱水　 70 %乙醇 6～ 2 4 h　 80 %乙醇 6～ 2 4 h　95%乙醇 1.5h　 10 0 %乙醇 1h。2 .2　氯仿透明 12～ 2 4 h。2 .3　浸蜡　…     

Practical aspects of planning, building, and interpreting tissue microarrays: The Cooperative Prostate Cancer Tissue Resource experience

This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.     

Fast quantification of immunohistochemistry tissue microarrays in lung carcinoma

Tissue microarrays (TMAs) are an effective tool for high-throughput molecular analysis of tissues to help identify new diagnostic and prognostic markers and targets in human cancers. We have developed a fully automated method for rapid, continuous and quantitative analysis of TMAs based on immunohistochemistry. The method deals with complex and varying tissue architectures, segments tumour cells from normal cells, conducts cell compartmentalisation, identifies nuclei and cytoplasm and produces three different continuous measurements of marker expression levels within tumour cell nuclei, tumour cell cytoplasm and total tumour cell protein expression. We have demonstrated this method using three independent protein markers (BAK, BAX and a novel biomarker, named KS) over 7 TMAs, involving 2 BAK stained TMAs with 229 tumour tissue cores, 2 BAX stained TMAs with 229 tumour tissue cores and 3 KS stained TMAs with 373 tumour cores of lung carcinomas. We validated the automated method, showing that the automated scoring is significantly correlated with the pathologist-based scoring.     

Construction of a tissue microarray with two millimeters cores of endometrioid endometrial cancer: factors affecting the quality of the recipient block

The tissue microarray (TMA) method currently is not used to render a primary diagnosis of cancer, but its scientific value has been proved in studies of various cancer types. TMA technology still is not used often for uterine tumors, however. We investigated the repeatability of histological diagnosis of endometrioid endometrial cancer (EEC) using conventional histology and TMA using 2 mm cores. We examined EEC tissues from 171 patients. Formalin fixed, paraffin embedded tissue donor blocks from EEC specimens were selected and examined histologically. Duplicate 2 mm tissue cores were inserted into a TMA recipient block. EEC tissues were examined as hematoxylin-eosin stained sections from the TMAs. EEC tissue was identified in the TMAs in 158 cases (92.4%) and not found in 13 cases (7.6%). On the TMA slides, both EEC positive cores were identified in 129 cases (75.4%), but only one core in 29 cases (17.0%). Among 342 biopsies of the donor blocks (each case in duplicate), EEC was found in 287 cases (83.9%) using the TMA: 124/146 (84.9%) with superficial infiltration, 153/178 (86.0%) with deep myometrial infiltration, and 10/18 (55.6%) without myometrial infiltration. We concluded that two 2 mm tissue cores from a biopsy of a donor block inserted into a TMA recipient block were sufficient to diagnose EEC in more than 90% of cases. EEC was identified in the TMAs with similar frequency with respect to superficial and deep myometrial infiltration. Cases without myometrial infiltration were identified less often.     

Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization

Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.     

New tissue microarray technology for analyses of gene expression in frozen pathological samples

Tissue microarrays (TMAs) are widely used to analyze gene expression in multiple pathological samples on a single slide. Currently, most TMA slides are made by coring paraffin-embedded tissues and arraying them into a paraffin block, from which TMA sections are cut. However paraffin-based TMA technology may not be compatible with frozen clinical tissue samples, which have a higher quality of RNAs and proteins for preparing TMAs than paraffin-embedded tissue samples. In this study, we developed an alternative TMA technology that is applicable to a broader range of frozen tissue samples. Our method takes advantage of a newly developed array recipient block that can be used to array small tissue cores. After arraying tissue cores, the tissue block can be immediately sectioned on a cryostat microtome to make TMA slides. TMAs made using this method have well-defined array configurations and good tissue/cell morphology. Immunohistochemistry and in situ hybridization study also revealed well-preserved proteins and mRNAs on TMA slides. Our method significantly simplifies TMA preparation and assembly when frozen pathological tissues are used. Our technology provides an alternative tool for creating high-quality TMAs for the general research community to study gene expressions in pathological samples.     

Chondrosarcoma of the spine: A rare case with unusual presentation

Background

TMAs are becoming a useful tool for research and quality control methods, mostly for immunohistochemistry and in situ hybridization.

Methods

A new technique that allows building TMA blocks with more than 300 tissue cores without using a recipient paraffin block for the tissue cores and without using a commercial TMA builder instrument is described. This technique is based on the construction of TMA needles modifying conventional hypodermic needles to punch tissue cores from donor blocks, which are attached by double-side adhesive tape on a computer-generated paper grid used to align the cores on the block mould, which is filled with liquid paraffin.

Results

More than two hundred TMA blocks were constructed using this method, utilized in immunohistochemistry and histochemistry as positive and negative controls and also in research.

Conclusion

This technique has the following advantages: it is easy to reproduce, affordable, quick and creates uniform blocks with more than 300 cores aligned, adherent and easy to cut, with negligible losses during cutting and immunohistochemistry and in situ hybridization procedures.     

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces.     

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a ‘donor’ block into a ‘recipient’ block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.     

Antibody-based tissue profiling as a tool for clinical proteomics

Here, we show a strategy for high-throughput antibody-based tissue profiling with the aim to create an atlas of protein expression patterns in normal human tissues and cancer tissues representing the 20 most prevalent cancer types. A set of standardized tissue microarrays (TMAs) was produced to allow for rapid screening of a multitude of different cells and tissues using immunohistochemistry. Eight TMA blocks were produced containing 48 different normal human tissues in triplicate and cancer tissue from 216 individually different tumors in duplicate. Sections from these blocks were immunohistochemically stained using five commercial and five in-house generated antibodies. Digital images for annotation of expression profiles were generated using a semiautomated approach. Five hundred seventy-six images and annotation data corresponding to a total of 30 Gbytes of data were collected for each antibody. The data presented here suggest that antibody-based profiling of protein expression in tissues can be used as a valuable tool in clinical proteomics.     

Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.