胚胎及生后不同发育时期昆明小鼠端脑的发育及β-APP的表达

研究胚胎及生后不同时期昆明小鼠端脑的组织学发生以及β淀粉样前体蛋白(β-amyloid precursor protein,β-APP)在其发育过程中的表达。方法取胚胎7.5、9.5d小鼠全胚,胚胎11.5、13.5、15.5d胎鼠头部,胚胎17.5d、生后1d、生后7d、生后14d以及成熟昆明小鼠的脑组织进行石蜡包埋、连续冠状及矢状切片,并进行HE染色、尼氏染色以及β-APP的免疫组化染色。结果⑴小鼠胚胎9.5d前神经管孔封闭,各脑泡结构分化明显,但尚未完全分化完成,胚胎11.5d已可分出明显5个脑泡,即端脑、间脑、中脑、后脑和末脑,之后各脑区按照不同的时空顺序发育成熟。⑵APP在小鼠胚胎7.5d有极少量表达,后表达逐步升高,17.5d达到高峰,主要表达在端脑皮质和海马;生后表达逐渐降低,直到成年组可见较广泛的表达。结论小鼠脑发育经历了复杂的变化过程,各脑区的发育呈不同时间空间变化,β-APP可能参与小鼠脑发育过程。     

小鼠MCMV先天感染模型的构建及神经系统感染状况的观察

目的 建立小鼠巨细胞病毒(MCMV)先天感染模型,并观察其脑组织的病理变化及感染状况.方法 取孕11～13.5d的BALB/c鼠,模型组羊膜腔微量接种K181毒株(病毒量1×103 PFU),对照组注射DMEM培养液.单独饲养5d后处死孕鼠,剖宫取出胎鼠,麻醉处死,取胎鼠脑组织制作冰冻切片.胎脑组织切片常规HE染色,光镜下观察病理学变化;采用免疫酶组化及免疫荧光法检测脑组织中的MCMV早期抗原.结果 感染组胎鼠存活率为71.9％.与对照组相比,羊膜腔内接种MCMV病毒对胎鼠存活率、吸收胎率、死胎率及胎鼠头部重量无明显影响,但可致胎鼠体重降低.感染组胎鼠脑组织出现明显的病理变化.在宫内感染组,免疫酶学组化法发现脑室区、脑室管膜下区、大脑皮质和海马区可见病毒感染细胞;免疫荧光法观察到的主要感染部位与免疫酶组化法基本一致.结论 通过羊膜腔内注射MCMV病毒成功建立MCMV先天感染小鼠模型,为研究MCMV先天感染致脑发育异常机制提供合适的整体研究模型.     

Wnt5a在雌性小鼠胚胎生殖管道的时空表达

目的 探讨Wnt5a在早期雌性生殖管道发育过程中的表达。 方法 正常孕15.5d、17.5d、19.5d和21.5d的小鼠胚胎,PCR鉴定性别,取雌性胎鼠石蜡包埋,连续横断切片,每4张行1张HE染色,1张白片待用。HE染色后获取连续切片图像,根据雌性胚鼠生殖管道解剖走形,识别中肾旁管/子宫解剖定位。选取含有中段中肾旁管/子宫的白片,用SABC免疫组织化学方法,检测Wnt5a在不同发育时期雌性生殖管道的表达。 结果 获取含雌性生殖管道的鼠胚切片。同时检测到Wnt5a在孕15.5d到21.5d的雌性鼠胚中段中肾旁管/子宫上皮细胞、间质细胞均有表达,随着鼠胚发育,Wnt5a在间质细胞表达不断增强,差异在统计学上有极显著意义（P <0.01）。 结论 Wnt5a在雌性小鼠生殖管道形成过程中发挥作用,可能是诱导子宫发育及分化的关键因子。     

塑料包埋结合四环素荧光标记制作不脱钙骨组织切片的实验研究

目的探讨塑料包埋技术结合四环素荧光标记制作不脱钙骨组织切片的关键步骤及应用。方法选取四环素荧光标记的兔股骨进行塑料包埋,Leica SP 1600切片机切片,荧光显微镜观察后用苦味酸品红染色,并与常规石蜡包埋切片相比较。结果四环素荧光标记不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、植入体及周围骨组织的整合程度,与常规石蜡包埋相比,染色层次分明,组织移位形变小。结论应用塑料包埋技术制作四环素荧光标记不脱钙骨组织切片,有利于行骨形态计量分析以及植入体与骨整合的研究。     

Research on plastic embedding combined with tetracycline fluorescence labeling in undecalcified bone tissue

目的 探讨塑料包埋技术结合四环素荧光标记制作不脱钙骨组织切片的关键步骤及应用.方法 选取四环素荧光标记的兔股骨进行塑料包埋,Leica SP 1600切片机切片,荧光显微镜观察后用苦味酸品红染色,并与常规石蜡包埋切片相比较.结果 四环素荧光标记不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、植入体及周围骨组织的整合程度,与常规石蜡包埋相比,染色层次分明,组织移位形变小.结论 应用塑料包埋技术制作四环素荧光标记不脱钙骨组织切片,有利于行骨形态计量分析以及植入体与骨整合的研究.     

塑料包埋技术在骨组织研究中的应用

目的研究塑料包埋技术制作的不脱钙硬组织切片,确定制作的关键步骤及用于骨组织研究的优点。方法选取狗胫骨节段或带有金属种植体的骨材料塑料包埋,LeicaSP1600切片机（德国）切片,用苦味酸品红染色观察,并与常规石蜡包埋相比较。结果不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、多孔材料及种植体周围骨组织的整合程度。与常规石蜡包埋相比,染色层次分明,组织移位形变小。结论应用规范塑料包埋技术制作不脱钙硬组织切片,更有利于行骨形态计量分析以及材料与骨整合的研究。     

塑料包埋技术在骨组织研究中的应用

目的 研究塑料包埋技术制作的不脱钙硬组织切片,确定制作的关键步骤及用于骨组织研究的优点.方法 选取狗胫骨节段或带有金属种植体的骨材料塑料包埋,LeicaSP1600切片机(德国)切片,用苦味酸品红染色观察,并与常规石蜡包埋相比较.结果 不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、多孔材料及种植体周围骨组织的整合程度.与常规石蜡包埋相比,染色层次分明,组织移位形变小.结论 应用规范塑料包埋技术制作不脱钙硬组织切片,更有利于行骨形态计量分析以及材料与骨整合的研究.     

影响淋巴结HE制片因素的探讨

淋巴瘤因其组织结构致密、细胞丰富等特点,使组织在脱水、透明、浸蜡、切片、染色等方面都受到一定的影响,导致病理诊断困难。作为一名合格的病理医师,要求懂得组织的石蜡包埋和HE染色的机制,熟练掌握石蜡切片和HE染色技术的操作并能够制出一张优质的病理玻片标本。本实验从石蜡切片和HE染色的机制出发,重点探讨淋巴结HE制片的注意事项。     

小鼠肾脏发育中凋亡细胞的超微结构变化

1　材料和方法1 1　动物取昆明小鼠 ,雌雄比例 3∶1同窝饲养 ,以雌鼠阴道精栓出现为第 1日计算胎鼠胚龄。部分孕鼠分笼饲养 2 0日后 ,每天早八时观察分娩情况 ,新生仔鼠按生后计算日龄。选取 1 4、1 8日胎鼠各 4只 ,生后 1、7、1 4日龄仔鼠各 4只为观察对象。1 2　光镜和电镜标本制作　在冷操作条件下切开胎鼠和仔鼠腹部 ,取左肾于 2 5 %戊二醛内固定。胎鼠做全肾树脂包埋 ,仔鼠肾脏经肾门做矢状面切开 ,皮质和髓质分别进行树脂包埋。ＬＫＢ Ｖ型超薄切片机半薄连续切片 ,甲苯胺蓝染色 ,按Ｋｏｓｅｋｉ判断光镜下凋亡细胞后制超薄切片 ,…     

胎儿胰腺的显微形态学观察

目的观察胎儿胰腺的结构特点,为胎胰临床应用提供资料.方法取30例21～41W胎儿胰腺,利用超薄切片及透射电镜观察和石蜡包埋切片HE染色、SP免疫组化方法进行染色.结果胎儿胰腺内分泌部随胎龄增加而相对减少;外分泌部逐渐增加.A、B细胞超微结构在21～41W无明显变化.结论 25～35w胎儿更适合临床应用.     

正丁醇联合松节油替代二甲苯用于石蜡包埋

目的 寻找一种能够替代二甲苯透明剂的组织石蜡包埋方法。 方法 使用正丁醇和松节油的混合物替代常规石蜡包埋过程中无水乙醇、二甲苯的作用,收取卷茎蓼干预的多发性脑梗模型大鼠的脑、肾、胃、肝、十二指肠组织进行石蜡包埋,最终根据石蜡切片的效果、HE染色以及免疫组织化学结果对这种新型脱水程序做出评价。 结果 正丁醇和松节油的混合物可以替代常规石蜡包埋程序中无水乙醇的脱水、二甲苯的透明作用。经正丁醇、松节油混合物处理后的组织切片流畅,组织无变脆、变硬;HE染色后,新型脱水处理组织的细胞核、细胞质分明,组织的着色度、色泽、透明度与常规脱水程序无差异;通过大鼠不同组织做免疫组织化学染色,经对比结果与常规包埋组织免疫组织化学染色无差异。 结论 正丁醇联合松节油用于组织脱水既能够避免二甲苯对人的毒性作用,还可以减少无水酒精过度脱水对组织的破坏。     

Immunohistochemical staining of plastic embedded bone marrow trephine biopsy specimens after microwave heating.

AIMS--To investigate (1) whether adequate immunohistochemical staining can be achieved on sections cut from plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer; and (2) whether this immunohistochemical staining is comparable with that achieved on routine sections cut from paraffin wax embedded trephine biopsy specimens after decalcification procedures. METHODS--Sixty five consecutive bone marrow trephine biopsy specimens of more than 1 cm in length were divided transversely into two equal parts. One part was processed in paraffin wax followed by decalcification. The other part was embedded in the epoxyresin Polarbed 812 followed by the cutting of 1 micron sections. Both parts underwent immunohistochemical staining by an identical panel of antibodies. With Polarbed 812 plastic embedded sections, microwave heating in citrate buffer was undertaken before the application of antisera. RESULTS--On sections cut from plastic embedded material, immunohistochemical staining was generally satisfactory, easy to interpret and comparable with that achieved with paraffin wax embedded material. Exceptions were antibodies to neutrophil elastase and CD61 where immunostaining was consistently negative on plastic embedded sections. Immunohistochemical staining for CD20 was consistently more reliable on plastic embedded sections. CONCLUSIONS--The results provide evidence that, with few exceptions, satisfactory immunohistochemical staining is possible on plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer. This, combined with the advantage of superior cellular morphology with semi-thin (1 micron) sections of plastic embedded material, make such embedding procedures the preferred method for the processing of bone marrow trephine biopsy specimens.     

Plastic embedding of bone marrow trephine biopsies for routine immunohistochemistry and diagnosis: our developments,updates and experiences over 20 years

Bone marrow trephine specimens are routinely examined for the histological investigation and diagnosis of lymphoma and other disorders. To achieve this, biopsies are usually fixed in formalin and embedded in paraffin wax for subsequent tinctorial and, often, immunohistochemical staining. However, in this review the authors report the historical developments of immunohistochemical staining on plastic sections, and, in particular, our own developments and updates during the last 20 years of using a plastic embedding procedure for the routine reporting of over 50,000 bone marrow trephines. Many evolutionary changes during this period have occurred to provide a simple technique for the successful and excellent demonstration of numerous cellular antigens. While the volume of work and experience relating to immunohistochemistry on plastic-embedded tissue is, the authors we believe, unique, the review also present why the current procedure may revert to use of paraffin wax in the future.     

球旁细胞光学显微镜下4种染色法结果观察

目的 探讨显示小鼠球旁细胞的适宜染色方法,更好地为教学和科研服务。 方法 取 20 只小鼠肾组织做石蜡切片,随机分为 4 组,分别用HE染色、高碘酸-Schiff 氏反应染色、改良结晶紫染色和免疫组织化学染色。 结果 HE染色和高碘酸-Schiff 氏反应染色不能显示肾球旁细胞;改良结晶紫染色和免疫组织化学均能较好地显示肾球旁细胞（胞质内有特征性的被染色的分泌颗粒）。 结论 改良结晶紫染色和免疫组织化学染色对球旁细胞有较好的染色效果,但改良的结晶紫染色更为简便、经济、稳定。     

Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

AIMS--To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS--Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS--In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS--These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied.     

Delineation of the healthy rabbit heart by immunohistochemistry – A technical note

Heart failure poses a big health problem and may result from obesity, smoking, alcohol and/or growing age. Studying pathological heart tissue demands accurate histological and immunohistochemical stainings in animal models, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy heart stainings and labeling are required, in order to provide a reference for the pathological situation. Heart and brain tissue of a healthy rabbit were collected, and different histological key steps were compared, such as paraffin embedding after formalin fixation versus cryopreservation; an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or a chromogenic with a fluorescent detection system, respectively. Using serial sections, we stained the same morphological structure with classic approaches (HE, Masson Goldner Trichrome (GT) and Elastica van Gieson (EL)) and with different markers, including collagen I, collagen III, fibronectin, α-SMA, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, and ki67 for proliferating cells. Differences between conditions were quantitatively assessed by measuring the color intensity. Generally, cryosections exhibited a more prominent signal intensity in immunohistochemically labeled sections than in paraffin sections, but the strong staining was slurry, which sometimes impeded proper identification of morphological structures, particularly at higher magnifications. In addition, the advantage of an AR step was observed when compared to the condition without AR, where signal intensities were significantly lower. Different stainings of the heart arteries and the myocardium revealed a clear distribution of extracellular matrix components, with prominent collagen III in the artery wall, but an absence of collagen III in the myocardium. Moreover, paraffin-embedded sections provided more distinct structures compared to cryosections after collagen III, ki67, fibronectin, and α-SMA labeling. As for the Purkinje cells that were depicted in the heart and the cerebellum (Purkinje neurons), we found GT staining most suitable to depict them in the heart, while HE as well as EL staining was ideal to depict Purkinje neurons in the cerebellum. In sum, we provide useful reference images with different stainings for researchers using the rabbit heart or brain model. Such images can help to decide which of the immunohistochemical protocols are valuable to reach a specific aim. Recommendations are given for the best visualization of the target structures and specific (immunohistochemical) staining.     

How the fixation-embedding protocol affects the specificity and efficiency of immunocytochemical stains for gonadotropin subunits

This report describes a study designed to test factors that may affect the efficiency and specificity of stains for gonadotropins. These include chemical or freeze-fixation and dehydration, heat polymerization of the plastic embedding compound, dehydration in organic solvents, and etching. Specifically, postembedding stains for LH or FSH subunits were applied to 1-μm sections of (1) Araldite-embedded pituitaries that were either chemically fixed and dehydrated or freeze-fixed and freeze-dried; (2) Aldehyde-fixed pituitaries that were dehydrated in water-soluble glycol methacrylate (GMA) and embedded in GMA at 4°C; and (3) p-formaldehyde-fixed pituitaries that were embedded in paraffin. A fourth group of pituitaries was dispersed and grown in monolayers for 1–3 days. These were stained following glutaraldehyde fixation. The optimal dilution of the primary antisera varied with the protocol; however, the percentage of cells staining for beta subunits did not change. In contrast, postembedding stains showed that alpha subunit reactivity is masked or destroyed in pituitaries that are fixed in glutaraldehyde and embedded in Araldite. Alpha chain reactivity was detected (in 14% of cells) either after freeze-fixation and freeze-drying followed by Araldite embedding, or after 4% paraformaldehyde fixation and GMA embedding (in 17% of cells). Staining in paraffin-embedded pituitaries was seen in only 10% of the cells. Preembedding stains for alpha chains were strikingly sensitive, however, and immunoreactivity was seen in 18–26% of the population of monolayer cells. Thus, whereas the percentages of cells staining for beta subunits do not change following the use of most of the fixation and embedding protocols, alpha chain reactivity is destroyed by all but the mildest. These findings show that one can control or improve the specificity of the stains for LH and FSH by the fixation-embedding protocol.     

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only approximately 30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.