一种用于DAPI染色的方法Steedman＇s wax包埋切片法

以植物的胚珠和子房为实验材料,介绍一种用Steedrnan‘s wax包埋对组织切片中的细胞核进行DAPI染色的方法。Steedrnan‘s wax作为一种低熔点多酯蜡,具有与石蜡相似的性质,切片方法同常规石蜡切片,适合于切成厚度大于5μm的连续切片。Steedrnan‘s wax包埋的切片能成功地进行DAPI染色。与用压片法和Technovit 7100或GMA包埋切片法进行的DAPI染色相比,用Steedrnan‘s wax包埋切片法进行的DAPI染色具有廉价、操作简便、可进行连续切片、图象清晰等优点,特别在植物细胞程序化死亡(PCD)的研究中及细胞核DNA含量测定方面,有着较大的应用价值和潜能。     

一种用于DAPI染色的方法--Steedman's wax包埋切片法

以植物的胚珠和子房为实验材料,介绍一种用Steedman's wax 包埋对组织切片中的细胞核进行DAPI染色的方法.Steedman's wax 作为一种低熔点多酯蜡,具有与石蜡相似的性质,切片方法同常规石蜡切片,适合于切成厚度大于5 μm的连续切片.Steedman's wax包埋的切片能成功地进行DAPI染色.与用压片法和Technovit 7100或GMA包埋切片法进行的DAPI染色相比,用Steedman's wax 包埋切片法进行的DAPI染色具有廉价、操作简便、可进行连续切片、图象清晰等优点,特别在植物细胞程序化死亡(PCD)的研究中及细胞核DNA含量测定方面,有着较大的应用价值和潜能.     

一种简单而快捷的半薄切片脱树脂新方法

目前,在HE染色、特殊染色或免疫组织化学染色研究方面多采用普通石蜡包埋切片,其切片厚度可薄达4μm,若更薄的切片,切割有一定的难度.对于各种染色而言,越薄的切片,细胞与组织结构越清晰,染色效果越好.环氧树脂(Epon)包埋的组织块不仅用于透射电子显微镜超薄切片的制备,也可用于光镜半薄切片的制备[1].经典的脱树脂的方法是将半薄切片置于氢氧化钠的无水乙醇饱和溶液中浸泡24h,然后再充分水洗.该法耗时长,容易脱片,本实验中摸索出一种简单而快捷的脱树脂的方法,现介绍如下.     

褐飞虱若虫中肠凋亡细胞的检测

为揭示褐飞虱Niloparvata lugens Stl若虫在发育过程中中肠的凋亡细胞,使用末端脱氧核苷酸转移酶介导的dUTP-生物素断端标记法(TUNEL)进行中肠组织切片检测,结果表明,1～5龄若虫中肠分别存在2%～5%的凋亡细胞。利用4′,6-二脒基-2-苯基吲哚二盐酸(DAPI)染色法检测表明,存在Ⅰ,Ⅱa和Ⅱb期凋亡的细胞核,其特征包括染色体浓缩、边缘化及细胞核碎裂。透射电子显微镜检测结果表明,早期凋亡的细胞呈现染色质浓缩、边缘化特征,晚期凋亡的细胞出现细胞核碎裂、形成凋亡小体及细胞质空泡化等。本研究揭示了在正常发育过程中褐飞虱若虫中肠有少量的细胞发生了凋亡。通过人工干预的方式调控中肠细胞的凋亡进程有可能使之成为防治该水稻害虫的新靶标。     

雷公藤甲素诱导小鼠急性肝损伤的形态学研究

目的:观察雷公藤甲素诱导小鼠急性肝损伤的形态学特征,为进一步研究雷公藤甲素肝毒性的病理特点和毒理机制提供基础。方法:昆明种小鼠以雷公藤甲素LD50剂量(0.8 mg/kg)水溶液灌胃,分别于给药12 h及24 h后取肝组织,制备石蜡切片、冰冻切片,行常规HE染色、PCNA染色、TUNEL染色及光镜观察。部分肝组织经戊二醛固定、制备超薄切片,行透射电镜下观察。PCNA及TUNEL染色结果采用图像分析软件进行定量分析及统计学处理。结果:0.8 mg/kg雷公藤甲素灌胃后12 h即可诱导肝组织炎细胞浸润、结构破坏、肝细胞坏死及代偿性增生。透射电镜下可见肝细胞内细胞骨架结构异常、细胞器大量脱落、自噬体明显增多。PCNA及TUNEL染色结果表明,雷公藤甲素可诱导肝细胞出现显著的增殖及凋亡。结论:雷公藤甲素可诱导肝组织炎性反应发生,同时伴随肝细胞凋亡、坏死及代偿性增生。推测肝细胞自噬性凋亡是雷公藤甲素诱导急性肝损伤的关键病理环节。     

HSV-2阴道感染并诱导小鼠阴道粘膜上皮细胞凋亡

最近的报道指出,某些病毒有诱导体外细胞凋亡的作用,借以限制病毒的扩散.为探讨HSV-2在体内诱导细胞凋亡的效果及其形态学特点,用HSV-2 333株感染小鼠阴道,于感染后不同天数处死动物,取其阴道,固定于10%中性福尔马林,TUNEL末端标记染色显示凋亡的细胞,光镜下进行原位观察.结果显示感染后的第1天粘膜上皮内即出现大量的凋亡细胞,第2天至11天凋亡细胞的数量及在上皮内的分布范围达最高水平.早期的凋亡细胞见于感染后所有标本,其核染色质形态及分布似正常细胞,但它被TUNEL标记染成棕黄色;晚期的凋亡细胞亦见于所有标本,其胞核缩小,染色质浓缩并在核周边集聚,核中心空化.载有凋亡细胞的上皮在阴道粘膜上分布很广,最广的可占全阴道上皮的2/3.同时可见HSV-2引起的上皮细胞坏死及疱疹形成,二者均由凋亡细胞包围.凋亡细胞不断地由上皮表面脱落至阴道,未见凋亡小体及吞噬现象.结果提示,HSV-2 333株阴道感染可同时诱导细胞坏死及凋亡,细胞凋亡可能在限制病毒产生子代及限制感染区域扩展起重要作用.     

四膜虫（Tetrahymena thermophila）核纤层的研究

采用非树脂包埋去包埋超薄切片电镜技术,并结合选择性生化抽提方法以及间接免疫荧光染色方法显示,在四膜虫细胞内存在典型的核纤层结构。蛋白分离纯化与免疫印迹法分析结果说明四膜虫细胞核纤层可能主要由分子量为６６ＫＤ的多肽构成。蛋白分离纯化结果表明四膜虫细胞的核纤层蛋白可能不如高等动物细胞的丰富。     

用于光学显微镜研究的塑料半薄切片

本文介绍了以环氧树脂为包埋介质的用于光学显微镜的塑料半薄切片的制备技术和部分实验结果。叙述了固定、脱水、渗透、包埋、聚合、切片、染色及封片各程序。作为对石蜡切片技术的补充和发展。塑料半薄切片能充分发挥光学显微镜的分辨能力,能观察到许多在石蜡切片上看不清或看不到的细胞内部结构,如:花粉的外粉壁,萌发孔;细胞的微核,液泡和液泡问的原生质丝等。可用同一包埋材料在半薄切片基础上进行超薄切片,所以半薄切片技术是一种把光学显微镜水平的研究和电子显微镜水平的研究联系在一起的一种过渡性技术。因此,它无论对植物学工作者或其它生物学工作者都是很有用的一项技术。     

原位缺口平移标记断裂DNA技术及其在检测流行性出血热尸检和实验动物组织中细胞凋亡和早期死亡的应用

原位缺口平移技术（ＩＳＮＴ）已被用于检测细胞核中ＤＮＡ断裂鉴别尸检组织中细胞的凋亡和坏死断裂、流行性出血热（ＥＨＦ）组织中存在散在单个细胞变性死亡和灶性梗死样坏死,前者带有细胞凋亡的特征。本文以ＥＨＦ肝脏和实验性病毒感染鼠脑组织为例,应用缺口平移法,在ＤＮＡ聚合酶或Ｋｌｅｎｏｗ酶的作用下,将地高辛标记的ｄＵＴＰ掺入合成到ＤＮＡ的断裂部位,通过碱性磷酸酶标抗地高辛抗体免疫组化法显示细胞ＤＮＡ的断裂,检测和鉴别细胞的凋亡和坏死。为分析死后解剖时间间隔及组织固定时间对该方法的影响,本文选用死后２～１４０ｈ尸检、经常规固定石蜡包埋后存放１０～３５年的标本和在１０％福尔马林固定了１０～３５年之后再进行常规处理的标本。实验时用蛋白酶Ｋ（ＰＫ）消化前后对比并分别在标记反应液中略去ＤＮＡ聚合酶作为阴性对照,用ＤＮＡ酶消化组织人为制造ＤＮＡ缺日作为阳性对照。结果发现,未经ＰＫ消化的组织,仅灶性肝细胞核ＩＳＮＴ标记阳性,经ＰＫ消化后,散在的带有凋亡特征的肝细胞胞核也出现阳性,灶性肝细胞胞核标记染色增强,但无论是蛋白酶消化与否,明确梗死样坏死的肝细胞均不被标记。结果还发现,死后２～２４ｈ内尸检组织和长时间（１０～３４年）存放的石     

肥胖、2型糖尿病过程中大鼠肝脏形态结构变化的比较

目的:探讨在肥胖、糖尿病过程中大鼠肝脏形态结构的变化.方法:Wistar雄性大鼠,随机分为:普通饮食组,高脂饮食组,高脂饮食+链脲佐菌素组.第0周随机抽取6只大鼠处死后取肝脏进行石蜡包埋,HE染色,制成切片.在饲养过程中选取不同时间点,各组随机处死2只大鼠,取肝脏进行石蜡包埋,HE染色,切片,镜下观察,比较各组差异.结果:与对照组相比,肥胖组大鼠肝脏组织中脂肪细胞数目增多、体积变大,随肥胖程度的增加而加剧,导致肝发生中、重度脂肪变性.肝细胞肿胀,肝窦间隙变窄,可见炎性细胞浸润增多,凋亡细胞增多.糖尿病组大鼠,肝脏组织中脂滴形成,出现大的脂囊.肝细胞出现细胞核固缩,凋亡细胞增多,有炎性细胞浸润,以淋巴细胞为主,未见巨噬细胞增多.血管壁硬化、玻璃样变性.结论:在肥胖/糖尿病发生、发展过中,大鼠肝脏中有脂肪的异位储存(肝脂肪样变性),并伴随有炎性细胞的浸润.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Use of nitrogen mustard N-oxide for the fixation of electron microscopic tissues

Summary Nitrogen mustard N-oxide was tried for the fixation of tissue for electron microscopy. A fixative consisting of 1% nitrogen mustard N-oxide, 1% glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 followed by 1% O_sO₄ buffered at pH 7.4 was found useful for the tissues examined: thyroid, anterior pituitary, adrenal gland and oviduct of mice.If the tissues are fixed and the sections are stained with uranyl acetate and lead acetate doubly, the follicle colloid, colloid droplets, and secretory granules containing thyroglobulin in the thyroid become higher in electron density. The cisterna of the maturing face of the Golgi apparatus, secretory granules, ribosomes, nucleolus and chromatin in the cells examined are extremely electron dense. Tubular elements of smooth endoplasmic reticulum in the adrenal cortical cell and microtubules in all the cells examined are also well preserved. The fixative containing nitrogen mustard N-oxide is useful also for cytochemistry. Using tissue fixed by this method and stained en bloc by uranyl acetate, the noradrenaline and adrenaline cells in the adrenal medulla are clearly distinguished by light microscopy.This study was supported by a grant from the Japan Educational Ministry     

A simple and direct pre-embedding technique for ultrastructure of scarce biological specimens

A simple and rapid technique for pre-embedding scarce biological specimens for Transmission electron microscopy (TEM) is reported. It is based on pre-embedding biological samples in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized with paraformaldehyde, glutaraldehyde, ammonium persulfate and Temed. Pre-embedding in BSA and BA offers several advantages over traditional pre-embedding techniques for TEM including the ability to visualize the sample and a more resistant matrix. This results in more reproducible and consistent analysis. It can be applied to tissues, cells, and subcellular structures handled as pellets or suspensions. In addition, use of the pre-embedding matrix for light microscopy is reported. The ability to pre-embed scarce biological specimens efficiently and reproducibly provides a valuable way to study and characterize cytological tissues such as biopsies or cystic and amniotic fluid cells.     

The effects of various fixatives on subsequent lectin binding to tissue sections

Summary The effects of ten fixation protocols on the subsequent binding of eight lectins to various mouse tissue sites have been systematically evaluated. The fixatives used were neutral and buffered formalin—saline, Bouin's fluid, 95% ethanol, Carnoy's fluid, calcium acetate—paraformaldehyde, and mercuric chloride both before and after removal of mercury pigment. These were compared with frozen sections of unfixed tissue and frozen sections post fixed in paraformaldehyde. Lectins used were PNA, DBA, SBA, BPA, UEA 1, GS I, GS II and MPA. Ethanol was found to be the superior fixative, closely followed by mercuric chloride. Paraformaldehyde was a poor fixative of both paraffin and frozen sections. It is recommended that, where a choice is possible, the fixation protocol appropriate to the particular lectin and tissue binding site is selected. Within certain limitations, formalin—saline proved an adequate fixative for the study of routine paraffin-processed tissue sections.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM). and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

Immunohistology on Formol-Sublimate Fixed and Paraplast Embedded Kidney Tissue with Comparison to Formalin Fixation and to Pre-Embedding Immunofluorescent Staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 μm Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.