肾活检冷冻组织石蜡制片的方法

肾空刺取出肾组织后,一般分为3部分,一部分制作成石蜡切片,一部分制作成冷冻组织切片,另一部分进行电镜检查。石蜡切片是肾活检病理诊断的最重要部分,若因切片未见肾小球或因其他原因导致制片失败,则不能进行肾活检病理诊断。而冷东肾组织经冻融后一般不能制备出满意的石蜡切片。     

肾穿标本的电镜样品制备方法

由于肾脏穿刺时取到的样品很小,穿到的部位又不易掌握,所以穿到的组织中常常没有肾小球.而肾小球的病变情况对肾脏疾病的诊断关系很大.在按常规进行电镜制样时,组织中有无肾小球或是否是肾脏组织,只有在包埋好切厚片观察时才能知道.而用本文介绍的方法既可以在标本包埋前就知道标本中有无肾小球或是别的组织,又可缩短制样时间.为临床医生的诊断,更快地提供有参考价值的资料.1 材料与方法1.1 试剂 8%多聚甲醛,2%戊二醛,Dodecenyl-succinic Anhydride,Methyl nadic anhydride,Epon812,DMP-30[2.46-tri(dimethylaminomethyl)phenol]0.16ml.1.2 方法 病人的肾穿标本用8%多聚甲醛(pH7.30)和2%戊二醛等量混合液(最终浓度多聚甲醛     

肾组织电镜包埋后银染色技术

银染色是肾脏组织学诊断的重要特殊染色技术,已广泛应用于肾脏疾病光镜水平鉴别诊断,然而电镜肾组织银染色技术的报道甚少,尤其是包埋后银染色技术国内还未见记载。我们参照国外的方法,开展电镜包埋后银染色技术获得满意结果。1 材料和方法1.1 取材人肾活检组织切成1mm~3大小,3.75%戊二醛4℃固定,0.1mol·L~(-1)pH7.4磷酸缓冲液漂洗3次,2%锇酸与3%亚     

肝移植穿刺活检组织快速石蜡制片

肝穿刺活检已成为临床诊断肝移植(OLT)术后并发症的主要手段之一.由于OLT术后急性排异等并发症常需要临床立即诊断和处理,因而需要在送检肝穿刺组织后尽可能早地发出病理诊断报告,但常规冷冻切片质量难以满足OLT组织病理学诊断的要求.为此,近几年来,我们逐步摸索出一种能达到与常规HE切片质量相似并明显优于常规冷冻切片的快速石蜡制片方法,为实施快速的OLT病理诊断提供了及时有效的技术支持.现将结果报道如下.     

石蜡切片组织透射电镜样品制备方法的比较分析

<正>透射电子显微镜(透射电镜)技术及石蜡包埋切片技术是临床病理诊断的重要手段[1-3]。一些穿刺或微小的组织,石蜡包埋切片数量有限,尤其某些特殊的微小结构仅能在少数切片上呈现,当需要做进一步的超微结构检测时,往往因切片不足而受限,因此,常需要在不影响原有结果的前提下,对切片进行二次检测。作者对石蜡切片组织透射电镜顶扣     

一种从石蜡包埋组织中快速提取DNA的方法

目的建立从甲醛固定石蜡包埋组织中高效、快速提取基因组DNA的方法。方法将一片10μm厚石蜡包埋大肠癌组织切片直接浸入150μl消化液中,56℃孵育0.5h、1h和2h,灭活蛋白酶K后离心取上清即为DNA提取物。琼脂糖凝胶电泳检测所提取DNA的片段分布,紫外分光光度法测定DNA的产量及纯度。以提取的DNA为模板,PCR扩增不同长度产物片段并进行产物的直接测序以评价提取方法。结果该提取方法能够获得较长片段的基因组DNA,从一片10μm厚石蜡切片中即可获得大约60μg的DNA,在0.5h消化条件下获取的模板DNA,即可实现对152bp、293bp、392bp和541bp等4种不同片段的100%扩增,1h消化组的PCR产物可直接用于DNA测序。结论建立了一种简单、快速地从甲醛固定石蜡包埋组织中提取DNA的方法,只需一步消化即可获得满足普通PCR及测序需要的基因组DNA。     

石蜡包埋乳腺癌组织单细胞悬液的制备

石蜡包埋乳腺癌组织单细胞悬液的制备董敬朋，陈艺华，张素娟乳腺癌是妇女常见的恶性肿瘤之一，而癌基因扩增、癌细胞ＤＮＡ倍体测定是判断乳腺癌患者预后的新指标。因此，我们建立了简便易行的用于ＤＮＡ倍体测定和癌基因检测技术的单细胞悬液制备方法。１材料和方法１．...     

石蜡包埋组织用于流式细胞光度术的单细胞悬液样品制备的研究

流式细胞光度术(Flow Cytometry)是一种快速定量分析细胞核酸含量的新技术,近年来已广泛用于肿瘤研究。单细胞悬液制备方法是应用该技术的重要环节。文献报道多采用新鲜肿瘤组织制备单细胞悬液。因此,不能结合恶性肿瘤病人     

电镜生物样品的平板包埋技术

为了完成下丘脑免疫组化、原位杂交电镜技术的研究课题,解决下丘脑核团定位难,免疫染色抗体穿透力差的问题,我们参考了国外的文献报道,探讨解决这两个问题的办法,我们知道要想获得免疫电镜、原     

内窥镜活检组织包埋方法的改良

内窥镜目前在我国各级医院较为普及 ,作内窥镜检查的人也不断增加 ,我院每年作病检的内窥镜的例次占全年总病检例次的 2 6 9%～ 32 5 % ,最多者为胃镜 ,由于取材方法及部位较固定的特点 ,我们对传统包埋方法进行了改良 ,使用镶嵌法及一片法包埋和切片 ,使用 8年多以来 ,效果较好 ,现介绍如下。1　材料与方法1.1　材料　纤维内窥镜取材为芝麻大小至米粒大小的活检组织。熔点为 5 6℃～ 5 8℃的生物切片石蜡。1.2　方法1.2 .1　包埋前先将融化的石蜡液倒入包埋框 ,然后以 0 5ｃｍ的间距 ,插入较薄的金属片 ,待石蜡冷却凝固后 ,即成为可使用…     

Rapid three-dimensional analysis of renal biopsy sections by low vacuum scanning electron microscopy

Renal biopsy paraffin sections were examined by low vacuum scanning electron microscopy (LVSEM) in the backscattered electron (BSE) mode, a novel method for rapid pathological analysis which allowed detailed and efficient three-dimensional observations of glomeruli. Renal samples that had been already diagnosed by light microscopy (LM) as exhibiting IgA nephropathy, minor glomerular abnormalities, and membranous glomerulonephritis (GN) were rapidly processed in the present study. Unstained paraffin sections of biopsy samples on glass slides were deparaffinized, stained with platinum blue (Pt-blue) or periodic acid silver-methenamine (PAM), and directly observed with a LVSEM. Overviews of whole sections and detailed observations of individual glomeruli were immediately performed at arbitrary magnifications between ×50 to ×18,000. Cut surface views and surface views of glomeruli were demonstrated at the same time. On Pt-blue-stained sections, podocytes, endothelia, mesangium, and glomerular basement membranes (GBMs) could be distinguished due to the different yields of BSE signals, and pathological features were investigated in every sample. The abnormal surface appearances of podocytes with foot processes and the varying thicknesses of GBM were revealed three-dimensionally, features difficult to observe under LM and transmission electron microscopy. PAM-positive GBM alterations in membranous GN were distinctly visualized through overlying cells without cell removal under LVSEM at high magnification. Not only prominent spike formation but also slight protrusions were clearly revealed in the side views of GBM. Crater-like or hole-like structures were shown in the en face views of GBM. Accordingly, LVSEM is expected to provide a novel approach to the pathological diagnosis of human glomerular diseases using conventional renal biopsy sections.     

An improved technic for processing aspiration biopsy for electron microscopy

This article describes a technic for processing aspiration biopsy samples for electron microscopy. Red blood cells present in these samples were separated from the tissue fragments with the use of a nylon sieve. The tissue fragments were pelleted and processed for electron microscopy in a routine manner. An analysis of 19 cases processed in this manner revealed that this separation technic is highly effective and results in a significant improvement in cellular yield.     

Application of low-vacuum scanning electron microscopy for renal biopsy specimens

Low-vacuum scanning electron microscopy (LV-SEM) has been developed which enables the observation of soft, moist, and electrically insulating materials without any pretreatment unlike conventional scanning electron microscopy, in which samples must be solid, dry and usually electrically conductive. The purpose of this study was to assess the usefulness of LV-SEM for renal biopsy specimens. We analyzed 20 renal biopsy samples obtained for diagnostic purposes. The sections were stained with periodic acid methenamine silver to enhance the contrast, and subsequently examined by LV-SEM. LV-SEM showed a precise and fine structure of the glomerulus in both formalin fixed paraffin and glutaraldehyde-osmium tetroxide-fixed epoxy resin sections up to 10,000-fold magnification. The spike formation on the basement membrane was clearly observed in the membranous nephropathy samples. Similarly to transmission electron microscopy, electron dense deposits were observed in the epoxy resin sections of the IgA nephropathy and membranous nephropathy samples. LV-SEM could accurately show various glomerular lesions at high magnification after a simple and rapid processing of the samples. We consider that this is a novel and useful diagnostic tool for renal pathologies.     

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.     

The formaldehyde-fixed and paraffin-embedded tissues for diagnostic transmission electron microscopy: a retrospective and prospective study

The fine structures of tissues processed routinely for light microscopy were studied retrospectively in 44 tissues and tumors and prospectively in 13 tissues and tumors. In the prospective study, we fixed tissues in an ample amount of fixatives, carefully avoiding crushed and air-dried portions, and processed them by five methods. The fine structures of the retrospective cases were mostly poor or passable, whereas those of the prospective cases were generally good. All formaldehyde-fixed tissues showed varied degrees of tissue extraction, notably of lipids, membranous structures, ribosomes, glycogen, and other loose cellular and intercellular matrix materials, that was related to the status and type of tissue as well as the kind and duration of fixation, dehydration, and tissue clearance. Paraffin embedding per se appeared to cause little alteration of the fine structure. Transmission electron microscopy of the paraffin-embedded tissue appeared most useful to identify infective agents, foreign particles, and densely packed organelles or structures, usually in the differential diagnosis of neoplasms. Although many factors are difficult to control, initial careful thin slicing, judicious selection, and fixation of the tissue (even by regular phosphate-buffered formaldehyde solution) can improve the fine structure of paraffin-embedded tissues and be useful in the direct correlation of light and electron microscopic findings.     

Rapid detection of cytomegalovirus using immune scanning electron microscopy

Monoclonal antibodies, specific for human cytomegalovirus, were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were then incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex-bead surface. No such binding of virus particles was detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.     

Rapid detection of cytomegalovirus using immune scanning electron microscopy

Monoclonal antibodies specific for human cytomegalovirus were conjugated to latex microspheres that were already labelled with rabbit anti-mouse immunoglobulin. The beads were incubated with serum or urine from patients, and then collected on a filter surface, which was analyzed in a scanning electron microscope. Size, immunological specificity, and relative quantity of virus particles were determined within 2 h after serum or urine collection by the visualization of virus particles specifically bound to the latex bead surface. No such binding of virus particles were detected in the various controls. This method was compared with conventional virus isolation by tissue culture. It enables identification of viruses within a few hours in different body fluids. Even without specific antibodies, the filtration method may permit the rapid detection of particles and the determination of their size in various body fluids.