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Sabine Bader Magdalena Zajac Thomas Friess Elisabeth Ruge Natascha Rieder Berthold Gierke Yvonne Heubach Marlene Thomas Michael Pawlak 《Molecular & cellular proteomics : MCP》2015,14(10):2775-2785
Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.Many human diseases are characterized by abnormalities in complex signaling pathways (1). The expression and activation status of proteins from these deregulated pathways has traditionally been analyzed using single marker techniques such as immunohistochemistry and Western blotting. Although these techniques have provided valuable information on the molecular abnormalities underlying human disease, they are labor intensive, have a low throughput, and often require high sample volume. Furthermore, techniques such as Western blotting are not applicable in the routine clinical setting. Miniaturized parallel immunoassay techniques have been developed in recent years and have played a pivotal role in biomarker discovery (2). Antibody arrays enable multiple potential disease markers to be investigated in a single sample in parallel (3). Beyond this, Reverse Phase Protein Arrays (RPPA)1 are sensitive high throughput tools that can quantify protein expression levels and activation status (posttranslational modifications such as phosphorylation) in multiple experimental samples simultaneously. The technique requires only minute amounts of samples, printed as lysate arrays onto slides, and hundreds of markers of interest can be investigated, array by array, in a miniaturized dot blot manner. Numerous reports have demonstrated that RPPA can be applied to various sources of cells and tissues to analyze protein profiles, signaling pathway networks, and for the identification of biomarkers (4–13). A recently published workshop report reviews the full potential and advances of RPPA for use in clinical, translational, and basic research (11).In oncology, the parallel profiling of multiple protein markers is particularly desirable to study tumor initiation and progression, to classify tumor disease states on the molecular level, and to discover and monitor biomarkers that can predict therapeutic response or tumor recurrence (14–16). The study of signaling response and analysis of pharmacodynamic (PD) markers upon treatment using in vitro and in vivo test systems (e.g. cell line or patient derived xenograft tumor models) is an established component of preclinical and early clinical drug development. These techniques can provide evidence of target pathway modulation for new therapeutic lead candidate compounds and provide valuable information on the drug mode of action (17), especially in the translational phase. Multiplex analyses of PD biomarkers by RPPA have been performed in vitro using cancer cell lines (18, 19) as well as in patient-derived tumor tissue and blood samples (20, 21) to assess response to treatment and target inhibition. A combination of RPPA signaling pathway mapping and functional PET imaging has recently been successfully evaluated in xenograft models as an early response PD marker for anti-cancer drug efficacy (13).Translating miniaturized multiple protein analysis platforms-such as RPPA - from preclinical to clinical applicability is highly desirable; however, issues such as the limited amount of available clinical samples and tumor heterogeneity must first be addressed. Furthermore, most studies of RPPA in tumor tissue to date have been conducted using proteins extracted from fresh-frozen (FF) tissue specimens; whereas, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation used in clinical pathology laboratories. FFPE yields excellent tissue architecture for histological assessment and enables analysis of individual proteins in situ by techniques such as immunohistochemistry. However, formalin fixation leads to extensive protein–protein and protein–nucleic acid cross-linking (22), which can hamper protein extraction and reduce both the overall yield of extracted protein and the profile of proteins detectable by proteomic techniques (23, 24). Furthermore, formalin-induced cross-linking induces conformational changes in protein structure that can alter the immunoreactivity of some proteins in situ by hiding or altering peptide epitopes (25, 26). Such artifacts are absent from snap-frozen tissue; therefore, protein profiles obtained from FF tissue are likely to reflect the in vivo biology of the tumor more closely. However, FF tumor tissue is not widely available because it is costly to collect and maintain in the clinical setting. FFPE tissue samples are routinely archived by nearly every hospital and offer a unique opportunity to study thousands of samples retrospectively with extensive clinical records and follow-up information.Several groups have now established protocols for retrieving cross-linked proteins from fixed tissues (27–33). These methods are mainly based on the use of concentrated ionic detergents and high temperature protocols closely related to the antigen retrieval methods developed for immunohistochemistry. These studies show that obtaining nondegraded, full-length proteins from FFPE tissues for multiplex analyses is feasible (27–33). More recently, protein extraction techniques optimized for fixed samples have been used to successfully conduct RPPA using FFPE tissue biopsies from different cancer types (34–40). Guo et al. systematically investigated several protein extraction methods and demonstrated that RPPA of FFPE materials is feasible, reproducible and can generate biologically relevant protein profiles (41). Other studies have confirmed the validity of this approach and shown that data generated from RPPA analyses of FFPE tissue demonstrate good concordance with traditional immunohistochemistry markers such as HER2 protein in breast cancer (34, 40). However, to date, analyses have been performed only for a limited set of protein markers.To evaluate whether analysis of a broader panel of protein markers is feasible and generates meaningful data from FFPE tumor tissue sections, we conducted RPPA on matched samples of FF and FFPE tissues using a set of 300 markers, the largest panel reported to date. Our aim was to identify markers that performed similarly when comparing the protein profiles measured in protein extracts from matched FF and FFPE tissue, using RPPA assays established for use in frozen tissues. Correlating selected markers and assays in such a way should qualify RPPA for further use with FFPE tissues of clinical relevance, e.g. in PD marker studies. In this paper, we have specifically focused on the technical issues relevant for using the RPPA platform in a clinical setting, and did not address the biology of the test systems used in detail. However, the models used have been pre-characterized to identify key signaling parameters in context of targeted drug treatment (42). We conducted a systematic comparison of RPPA protein profiles in matched FF and FFPE tumor tissues resected from three different xenograft models of human cancer, each treated with targeted therapeutic antibodies that have previously been shown to achieve tumor growth inhibition. Furthermore, we investigated the effect of targeted drug treatment on protein expression and activation status, and the concordance of matched FF and FFPE tissue RPPA profiles. Finally, with one of the applied tumor models, we compared a set of protein profiles measured with two different multiple assay platforms - the RPPA and the Luminex Bio-Plex system, and discuss their relevance with respect to the analysis of FFPE tissue. 相似文献
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Marshall S. Scicchitano Deidre A. Dalmas Rogely W. Boyce Heath C. Thomas Kendall S. Frazier 《The journal of histochemistry and cytochemistry》2009,57(9):849-860
Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)–embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT–embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis. (J Histochem Cytochem 57:849–860, 2009) 相似文献
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A. Sally Davis Anke Richter Steven Becker Jenna E. Moyer Aline Sandouk Jeff Skinner Jeffery K. Taubenberger 《The journal of histochemistry and cytochemistry》2014,62(6):405-423
Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ2 (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ2 data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraffin-embedded mouse lung tissue fixed in different fixatives. 相似文献
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Robert W. Sprung Jr. Jonathan W. C. Brock Jarred P. Tanksley Ming Li Mary Kay Washington Robbert J. C. Slebos Daniel C. Liebler 《Molecular & cellular proteomics : MCP》2009,8(8):1988-1998
Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 μg of protein yielded ∼400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. Although the status of posttranslational modifications was not examined in this study, archival samples displayed a modest increase in methionine oxidation, from ∼17% after one year of storage to ∼25% after 10 years. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery.Formalin-fixed paraffin-embedded (FFPE)1 tissue samples are routinely prepared during the pathological characterization of clinical specimens and are abundantly available in pathology archives worldwide. The fixation process yields clinically relevant samples that can be stored at ambient temperature and are suitable for pathological examination by light microscopy even after years in storage. Given the wealth of clinical data associated with specimens collected over a span of decades, such as patient treatment regimens and outcomes, FFPE tissue represents a potentially valuable resource for biomarker discovery through retrospective analysis (1, 2).However, fixation of tissue in formalin leads to significant cross-linking among proteins and other biomolecules, rendering the samples incompatible with many biochemical analyses. Immunohistochemical (IHC) analysis of FFPE tissue has been conducted since the 1970s using either proteolysis or protein denaturants to expose antigenic regions of proteins (3, 4). Since the 1990s, detection of antigens in FFPE tissue has been improved through the development of so-called antigen retrieval techniques (5, 6). These methods involve application of heat in the presence of any of a variety of buffers resulting in the cleavage of methylene bridges formed during the course of fixation (2).Despite their utilization for IHC analysis, FFPE tissue samples have been largely overlooked in proteomics studies, due to the assumption that tissue fixation would make proteomic analysis intractable. Recent work appears to refute this notion. In 2005, Hood et al. (7) first described the successful application of shotgun proteome analysis to FFPE tissue. Using laser capture microdissected cells and an optimized extraction method, hundreds of proteins were identified from a cancerous prostate lesion and benign prostate hyperplasia, thus opening the door to comparative proteomic analyses of FFPE tissue. Moreover, the same study showed that the numbers and identities of proteins observed were remarkably similar when applying the method to frozen and FFPE mouse liver, thus lending support to the use of FFPE tissue in biomarker discovery studies. Since the initial demonstration of its feasibility, FFPE tissues from diverse origins including breast, liver, kidney, lymphoma, and bone successfully have been subjected to proteomic analyses (8–14).Although this work suggests the feasibility of biomarker discovery from FFPE tissue, most of these previous studies have been performed on small amounts of material with one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The use of multidimensional peptide separations can extend the dynamic range of the LC-MS/MS analyses to detect lower abundance proteins. Recently, the use of capillary isotachophoresis as the first dimension in a multidimensional peptide separation strategy for analyzing FFPE tissue was described (8). In this study, thousands of proteins were identified out of <4 μg of digest from FFPE human liver sections. However, the apparatus used was an in-house, custom-designed system, not readily accessible to other laboratories. In several of these studies, proteins identified by a single peptide were accepted as valid identifications. Use of single peptide-based identifications elevates the probability of false positive protein identifications, and these identifications often constitute the majority of protein identifications (15).The equivalence of fresh/frozen and FFPE tissue proteomes is a critical issue in evaluating the suitability of employing FFPE tissues for biomarker discovery by comparative proteomic analyses. Hood et al. (7) and Guo et al. (14) reported comparisons from analyses of paired fresh and frozen tissue specimens. Guo et al. (14) reported an apparent overlap of 83% in protein identifications between FFPE and frozen brain tissue specimens, whereas Hood et al. (7) did not report the degree of overlap, but found that FFPE mouse liver tissue yielded about 88% of the identifications determined for frozen mouse liver tissue. The majority of protein identifications in both studies were based on single peptide assignments. These investigations did not explicitly address the effect of formaldehyde-derived modifications on the inventories of identified peptides.An unexplored question with FFPE tissue specimens is the extent to which normal variability in fixation process and storage duration affect the proteomes observed. The duration of tissue fixation is not highly standardized and may vary from hours to several days. One of the most attractive features of FFPE specimens is the opportunity for retrospective biomarker discovery, but the effects of storage for many years on tissue proteomes remains unknown.Here, we address these questions through detailed comparative studies of the analysis of fresh frozen and FFPE tissues by LC-MS/MS-based shotgun proteomics. We used the same fresh tissue specimens to prepare both frozen and FFPE samples for paired comparisons. We evaluated conditions for tissue lysis and digestion and the effects of fixation time and storage duration on the number of protein IDs obtained during shotgun proteomic analysis of FFPE tissue. We also characterized the differences in peptides observed between fixed and frozen specimens in an effort to understand the effect of fixation from a practical biomarker discovery standpoint. Furthermore, we compared analyses of fresh frozen and FFPE colon adenoma tissue by multidimensional LC-MS/MS using gel-based isoelectric focusing of peptides (Fig. 1). The results demonstrate a remarkable overlap in the number and identities of proteins between the fixed and frozen tissue and indicate that variations in duration of fixation and storage have a minimal effect on protein inventories obtained by shotgun proteomic analysis. The data indicate essential equivalence between protein inventories obtained from fresh frozen and FFPE tissue specimens by shotgun proteomics and validate the use of FFPE tissue specimens for biomarker discovery.Open in a separate windowFig. 1.Strategy for multidimensional LC-MS/MS analysis of FFPE tissue. 相似文献
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Dimo Dietrich Ralf Lesche Reimo Tetzner Manuel Krispin J?rn Dietrich Wolfgang Haedicke Matthias Schuster Glen Kristiansen 《The journal of histochemistry and cytochemistry》2009,57(5):477-489
A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells. (J Histochem Cytochem 57:477–489, 2009) 相似文献
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Morgan H. McCoy Kristin Post Joyashree D. Sen Hsim Y. Chang Zijin Zhao Rong Fan Shaoxiong Chen Diane Leland Liang Cheng Jingmei Lin 《Journal of visualized experiments : JoVE》2014,(89)
It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies. 相似文献
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Archival tissue specimens are commonly stored as formalin- fixed, paraffin wax-embedded blocks. Formalin fixation facilitates excellent morphological preservation, and the immunoreactivity of many antigens is preserved, but formalin- induced chemical cross-linking of proteins renders them insoluble and inaccessible to standard biochemical extraction and analytical methods. Thus, biochemical analysis of tissue components identified by histochemistry, with the advantage of long-term clinical follow-up, is precluded. We have applied cyanogen bromide cleavage, a technique used routinely for fragmenting proteins for sequencing experiments, to solubilize transferrin polypeptides and glyco-polypeptides from formalin- fixed, paraffin wax-embedded rat liver. Cyanogen bromide cleaves protein specifically at methionine residues, yielding a predictable array of polypeptide fragments. Subsequent oligosaccharide analysis of the transferrin glycopolypeptides by anion exchange chromatography confirmed that, in addition to successful release of polypeptide chains, sialylated oligosaccharide structures remained intact after cyanogen bromide cleavage. This approach may have wide applicability to a range of research interests in which correlation of tissue biochemistry with long-term follow-up is advantageous. 相似文献
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Zhefeng Xiao Guoqing Li Yongheng Chen Maoyu Li Fang Peng Cui Li Feng Li Yanhui Yu Yongmei Ouyang Zhiqiang Xiao Zhuchu Chen 《The journal of histochemistry and cytochemistry》2010,58(6):517-527
Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method. (J Histochem Cytochem 58:517–527, 2010) 相似文献
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应用多重PCR方法检测并鉴别石蜡包埋组织中的结核分枝杆菌复合体与非结核分枝杆菌DNA扩增片段类型 ,为结核分枝杆菌复合体感染与非结核分枝杆菌感染的病理学诊断提供一种补充的鉴别诊断方法。应用三对具有特异性的寡核苷酸引物 ,进行多重PCR扩增。这三对引物分别对应于分枝杆菌 6 5kD表面抗原、结核分枝杆菌插入序列IS6 1 1 0及人类β 珠蛋白基因的部分序列 ,其扩增产物分别为 3 83bp、1 2 3bp和 2 6 8bp。此种多重PCR方法检测的灵敏度为 0 6pg。经多重PCR扩增后进行凝胶电泳 ,结核分枝杆菌复合体 (结核分枝杆菌、牛型结核分枝杆菌、BCG)均可见 3 83bp、1 2 3bp片段 ,而非结核分枝杆菌 (鸟、龟、瘰疬、蟾蜍、堪萨斯、胞内、耻垢分枝杆菌 )仅见 3 83bp片段 (猿猴分枝杆菌与结核分枝杆菌复合体相同 )。与上述相比 ,分枝杆菌感染的临床标本分别增加了一条 2 6 8bp片段。对 2 0 9例临床初步诊断为淋巴结结核病人的石蜡包埋组织标本进行了多重PCR检测 ,1 93例病理诊断为淋巴结结核、结核性肉芽组织、结核性肉芽肿性炎症病人的标本 ,检测结果符合结核分枝杆菌复合体感… 相似文献
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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. 相似文献
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Punched-Out Agar Petri Plates for Systematic Fixation and Embedding of Multiple Small Tissue Samples
A 5% agar solution is poured into Petri dishes, jelled, and a pattern of cylindrical holes punched out according to the number and size of tissue samples to be processed. These punched-out Petri plates withstand neutral formalin, water, benzene, and paraffin immersion at 60 C; thus providing an adaptable container for processing small tissue samples. Reduced handling of the tissues, minimized risk of loss, and easy identification of samples by means of the map-like arrangement of the holes are among the virtues of such plates. An optimal fixative-volume: tissue-weight ratio can be attained by using wells of varying diameter and depth according to the sample size. 相似文献
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从取材于3只SIV感染治疗猴和4只SIV艾滋病模型猴治疗前后的11份石蜡包埋淋巴结活检组织中提成基因组DNA,分别用PCR和巢式PCR方法检测SIV病毒基因,PCR共检出10份阳性,巢式PCR检测11价样品均阳性,而同期采样的猴外周血标本病毒分离仅3份阳性,PCR扩增产物的特异性用限制性内切酶酶切反应得到证实。实验说明,从淋巴结组织中检测SIV的病毒基因较外周血病毒分离更能真实地反映病毒感染状况,本研究将对全面和正确评价艾滋病药吻和疫苗治疗效果提供帮助。 相似文献
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Srikanth Kudithipudi Denis Kusevic Sara Weirich Albert Jeltsch 《Journal of visualized experiments : JoVE》2014,(93)
Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs. 相似文献