三种不同方法固定的石蜡切片中RNA的分析

研究在 10 %中性福尔马林、丙酮、甲醇 氯仿 冰醋酸 3种方法固定的石蜡切片中提取RNA的质量和数量 .取 2 5 0g体重的Wistar大鼠的肾脏 ,分别采用 10 %中性福尔马林、丙酮、甲醇 氯仿 冰醋酸 3种方法固定 ,石蜡包埋 ,H E染色 ;采用RNA裂解液、TRIZOL试剂 2种方法提取切片RNA ,逆转录为cDNA ,采用普通PCR和SYBRGREEN 1定量PCR分析RNA质量和数量 .结果表明 ,3种固定方法都可保持组织良好的结构和形态 ;采用 2种提取方法 ,均可经RT PCR扩增出 180bp大鼠磷酸甘油醛脱氢酶 (G3PDH)、5 6 5bpβ肌动蛋白 (β actin)、10 0bp纤溶酶系活化剂抑制物 1(PAI 1) ;但采用RNA裂解液时 ,比TRIZOL试剂可提取更多的RNA .     

黄梁木维管组织细胞激光显微切割技术体系建立

黄梁木是华南地区重要的速生用材树种,其快速的次生生长主要取决于包括形成层细胞在内的维管组织细胞的发育进程。为了精准获取维管组织形成层细胞与高质量RNA,该研究以黄梁木幼苗为材料,通过石蜡切片与激光显微切割(LMD)结合的方法,成功获得形成层、木质部和韧皮部细胞。结果表明:优化石蜡切片流程,以卡诺/丙酮作为固定剂(丙酮浓度10%~50%),100%正丁醇作为脱水剂和透明剂,之后经过梯度渗蜡、包埋、切片、脱蜡后可获得形态完整的组织切片且保证了组织细胞RNA的完整性。调试LMD参数,放大倍数为10×~20×;激光能量为42~44;切割孔径为4~13;切割速度为16~25;最终获得的这3种细胞的RNA完整性(RIN)均大于5.0,即形成层细胞RIN=5.0,韧皮部细胞RIN=6.6和木质部细胞RIN=7.9,满足后续RNA-seq分析。该研究通过优化石蜡切片和LMD参数,成功建立了黄梁木幼苗茎段维管组织细胞的激光显微切割技术体系,为进一步揭示林木树种维管组织分裂与分化的调控机制奠定基础。     

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。     

应用激光显微切割技术检测单个结肠细胞的基因表达

目的：探讨检测单个结肠细胞的基因表达的方法。方法：应用激光显微切割技术(1aser micmdissection)从冰冻切片上将单个结肠细胞切下,提取总RNA,将RNA逆转录成cDNA,采用巢式逆转录聚合酶链反应(nested RT—PCR)检测mRNA的表达。结果：在显微镜下用紫外激光显微切割机,将单个结肠细胞成功切下,提取RNA后,逆转录成cDNA,经过巢式RT—PCR扩增后,扩增产物在琼脂糖凝胶上清晰可见。结论：联合应用激光显微切割和巢式RT—PCR可以检测单个结肠细胞的基因表达。     

激光显微切割技术用于子宫内膜异位组织DNA的提取及其完整性分析

目的：探索一套激光显微切割（LCM）分离子宫内膜异位症腺体细胞后提取微量DNA并进行完整性分析的操作流程。方法：分别对20例石蜡标本及20例冰冻标本进行LCM,收集切割后的腺体细胞;2组标本各取10例提取微量DNA,检测DNA浓度并通过PCR扩增进行验证;余20例标本分别进行全基因组扩增,检测产物浓度并利用8种常见管家基因作为引物通过PCR扩增进行验证,对比分析其结果。结果：石蜡标本与冰冻标本在LCM获取腺体细胞及提取微量DNA两个环节中均可获得满意效果;但经全基因组扩增后,石蜡标本无法保留完整DNA信息。结论：LCM获取子宫内膜异位症腺体细胞提取微量DNA是一种操作简单、结果稳定的方法,可作为日后子宫内膜异位症基因组研究的常规方法;冰冻切片相对石蜡切片,更能保留完整的DNA信息。     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

冰冻切片与石蜡切片对乳腺肿瘤的诊断价值研究

目的:分析和比较冰冻切片与石蜡切片对乳腺肿瘤的诊断价值。方法:选取480例新鲜乳腺标本,将其制成冰冻切片以及石蜡切片,根据诊断结果进行对比分析,评价乳腺肿瘤的冰冻切片与石蜡切片的对乳腺肿瘤的诊断价值。结果:经石蜡切片诊断乳腺良性肿瘤277例,占57.71%,良性肿瘤中以乳腺纤维瘤诊断居多;经石蜡切片诊断乳腺恶性肿瘤203例,占42.29%,以乳腺浸润性导管癌居多。冰冻切片诊断乳腺良性肿瘤279例,占58.13%;恶性肿瘤195例,占40.62%;延迟诊断6例,占1.25%。以石蜡切片诊断结果为金标准,冰冻切片诊断乳腺良性肿瘤的准确率为98.56%(273/277),诊断恶性肿瘤的准确率为95.07%(193/203),假阳性率为0.72%(2/277),假阴性率为2.96%(6/203),冰冻切片与石蜡切片诊断乳腺肿瘤的结果具有显著一致性,K值为0.965(P0.05)。结论:冰冻切片与石蜡切片诊断乳腺肿瘤的符合率较高,可作为术中快速病理检测的手段,但该种切片方式存在少量延迟诊断,多与术者操作经验有关,故术中应注重制片过程,提高冰冻切片质量。     

Trizol RNA提取法在雪貂组织的流感病毒H3N2定量PCR检测中优于RNeasy mini kit（Qiagen）柱子法

目的比较RNeasy mini kit（Qiagen）柱子法和Trizol法提取RNA在雪貂肺和肠组织H3N2 SYBRGreen PCR定量中的去干扰作用,从而找到适合该定量体系的RNA提取方法。方法比较Trizol法,RNeasy minikit柱子法,改良RNeasy mini kit柱子法1和改良RNeasy mini kit柱子法2提取肺肠组织RNA的质量,RNA逆转录成cDNA后,利用SYBR Green PCR方法检测样品中H3N2的载量,比较产物的特异性,综合评价RNA提取方法。结果 4种方法提取的肺和肠组织的RNA质量不相同,紫外分光光度仪测得RNeasy mini kit柱子法提取的RNA的A260/280低于1.8,其余3种方法的A 260/280在1.8～2.1之间,A 260/230只有Trizol法能达到2.0左右,其余3种方法均远低于2.0。电泳可见Trizol法提取的RNA有3条带：28 s、18 s和5 s,而RNeasy mini kit柱子法的RNA除了有28 s、18 s外,还有基因组DNA,改良RNeasy mini kit柱子法1和2提取的RNA只有28 s和18 s带。RNA逆转录后进行荧光定量PCR,从溶解曲线看,不论是鼻甲刮取物还是肺肠组织的样品模板,4种方法获得的样品与标准品均为单一峰溶解曲线峰,并且波峰位置重叠。而鼻甲刮取物定量的产物跑琼脂糖凝胶电泳均为单一的特异性条带,而肺肠组织的产物电泳发现：Trizol法获得的模板定量产物与标准品一致,为单一的特异性条带,而其它3种方法获得的模板则均有非特异性条带。结论鼻甲刮取物的病毒定量选用RNeasy mini kit柱子法提取定量结果与Trizol法一样可靠,说明对于简单样品该体系及引物非常适用,结果可信。对于组织样品肺和肠,Trizol法获得的样品定量结果比其它3种方法可靠。     

观察比较贝母类生物碱分布的3种切片方法

目的:建立一种能更清楚有效地观察贝母鳞茎中生物碱沉淀分布及含量的切片方法。方法:以新疆贝母为供试材料,利用石蜡切片法、徒手切片法及改良切片法制备植物鳞茎组织切片;切片经改良碘化铋钾处理,并通过Mikroskop Primo Star X2005显微镜观察和比较3种不同切片中生物碱沉淀分布。结果:改良切片法处理的切片中生物碱沉淀分布观察清晰,可用于定性及定量分析,并且实验过程中试剂对生物碱的影响较小,操作方法简便、快速、有效。结论:贝母鳞茎生物碱的观测适合采用改良切片法制片。     

成熟玉米秆组织结构切片方法的比较研究

于玉米成熟期选择健壮茎秆的基部第三节间为材料,采用徒手切片法、冰冻切片法、石蜡切片法、薄切片法等4种方法,比较不同方法的玉米茎秆组织结构切片质量,为研究玉米茎秆结构与其倒伏的关系奠定技术基础。结果表明:徒手切片法是获得成熟玉米秆组织结构切片较为方便、快速的方法,切片面积较大,适合大范围观察统计;冰冻切片法是获得成熟玉米秆组织结构较快的方法,切片面积较小,适合小范围观察;薄切片是获得高质量成熟玉米秆组织结构切片的最好方法,但切片面积太小,适合高倍数显微观察和小范围电镜扫描观察组织结构;石蜡切片不适合作为成熟玉米秆的组织切片方法。研究认为,徒手切片法是最适合成熟玉米秆组织结构观察研究的制片方法。     

The effect of different fixatives and length of fixation time on subsequent AgNOR staining for frozen and paraffin-embedded tissue sections

Summary The AgNOR technique has been used extensively in studies investigating the possibility that the numbers and appearances of the intranuclear structures stained are markers of malignancy. The method has the advantage of being applicable to many different types of histological material, including paraffin-embedded tissue. However, it has been suggested that the visualization of AgNORs is dependent on the type and time of fixation employed. This study set out to measure this effect with the following commonly-used fixatives: acetone, absolute ethanol, methanol, Carnoy's fluid, Bouin's fluid, 4% glutaraldehyde, 10% neutral buffered formalin and 10% formol-saline. Both frozen sections and blocks of fresh tonsil were fixed for varying times, the blocks of tissue then being processed routinely. With the frozen sections AgNORs were easier to discern than in sections of paraffin-embedded tissue, and more intranucleolar AgNORs were visible when alcoholic fixatives were used than with aldehyde fixation. The effects of different fixatives on AgNOR appearance in paraffin sections is, however, more complex. Despite the variation caused by different fixatives, AgNORs could be demonstrated adequately with all the fixatives studied. It is concluded that fixation is not a limitation to the study of AgNORs provided that the time and type of fixative is controlled.     

Fixation-dependent cytoplasmic false-positive staining with an immunoperoxidase method

Fixation-dependent nonspecific staining with the unlabeled immunoperoxidase (PAP) method was studied using paraffin-embedded human spleen sections fixed in various fixatives; the specific primary antiserum was omitted or nonimmunized normal rabbit serum was used. Strong cytoplasmic staining of polymorphonuclear leucocytes and macrophages was found after fixation in acetone, alcoholic formalin (94% alcohol) and absolute ethanol. This staining was mainly produced by the second layer of the PAP method. The most probable explanation of this phenomenon is nonspecific protein-immunoglobulin interaction as a result of alcoholic or acetone fixation of the sections. The present findings point to the importance of controls for each case under study to avoid false-positive interpretations.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.     

Lymphocyte membrane antigens in glycol methacrylate embedded tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Effect of Immunohistochemistry on Molecular Analysis of Tissue Samples: Implications for Microdissection Technologies

Laser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the 1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno–laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

Summary We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I,-III and-IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and-III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraftin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.