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。     

不同固定液和染色方法显示山羊子宫内肥大细胞效果的比较

目的探讨用不同固定液和染色方法对显示处于间情期山羊子宫肥大细胞的影响。方法用四种不同的固定方法,应用改良甲苯胺蓝（MTB）染色法和阿尔辛蓝-番红花红（AB-S）染色法显示处于间情期山羊子宫肥大细胞。结果山羊子宫组织采用Carnoy氏液固定,MTB和AB-S染色对所有的肥大细胞均可获得良好的染色反应,但10％中性福尔马林,4％多聚甲醛,Bouin氏液固定的组织仅有少量肥大细胞着染。结论MTB和AB-S染色法均是山羊子宫肥大细胞良好的染色方法。     

改良PAS染色法在急性肝损伤中的应用

目的：观察改良肝脏糖原PAS染色法,并观察肝脏糖原染色在急性肝损伤中的应用。方法：复制CCl4急性肝损伤模型,首次100％CCl43 mL／kg皮下注射,此后50％CCl4橄榄油溶液2 mL／kg每周2次共4次皮下注射,诱导大鼠急性肝损伤模型。计算大鼠肝体比;HE染色观察肝组织炎症病理;试剂盒检测血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、总胆红素（TBil）、白蛋白（Alb）。肝脏常规PAS染色与改良PAS染色观察肝糖原染色。结果：与正常组相比,模型组ALT、AST活性与TBil含量明显升高（P＜0．05）,Alb含量明显降低（P＜0．05）;HE染色示,模型组肝小叶结构排列紊乱,肝细胞脂肪变、气球样变明显。常规PAS染色,正常组肝组织PAS染色阳性占肝脏面积为32．38％±5．50％;与正常组相比,模型组肝组织PAS阳性染色明显减少（P＜0．01）,占肝脏面积为8．60％±3．34％。改良PAS染色提示,正常组肝脏可见大量PAS阳性染色,占肝脏面积为75．50％±9．02％;与正常组相比,模型组肝组织PAS阳性染色明显减少（P＜0．01）,占肝脏面积为17．61％±3．53％。在空白对照组与模型肝组织中,肝糖原改良PAS染色阳性率明显高于常规PAS染色法（P＜0．01）。改良PAS染色肝糖原阳性染色面积更真实反映急性肝损伤程度。结论：改良肝脏糖原PAS染色法有助于急性肝损伤程度评估。     

应用VAN-Clear与低甲醛的病理组织处理改良方法的评价

目的探索一种可靠、稳定、高效、环保、可替代传统病理组织处理与制片方法的改良方法。方法选取10只实验大鼠,每只大鼠选取14种组织,每个组织均匀切成3块,第一块采用4%中性甲醛固定(改良中性甲醛组),第二块采用4%多聚甲醛固定(改良多聚甲醛组),第三块采用4%中性甲醛固定(传统中性甲醛组);两个改良组的组织经相应固定液固定后均用流水及75%酒精祛除组织的固定液,然后进入梯度酒精及VAN-Clear进行脱水透明;传统中性甲醛组在固定后不进行祛除固定液处理而直接入梯度酒精及二甲苯进行脱水透明。对比3组实验的HE、Masson、PAS及免疫组织化学染色效果和HE染色评分;比较取材室及技术室的甲醛、二甲苯及噪声污染的数据。结果两个改良组与传统组的HE、Masson、PAS及免疫组织化学染色效果及HE染色评分基本一致,3组HE切片的优良率均为100%,优级率均大于85%;甲醛及二甲苯检测结果远远低于我国的最高容许浓度,噪声监测优于中国环境噪声容许的夜间噪声标准。结论采用VAN-Clear以及低甲醛的病理组织处理改良方法可靠、稳定、高效、环保,可替代传统病理组织处理方法。     

常用实验动物不同组织部位肥大细胞异质性的组织学特点比较

目的探讨并比较六种常用实验动物不同部位肥大细胞异质性的形态学特点。方法应用麻醉后处死的方法,取小鼠、大鼠、豚鼠、兔、犬和猴的皮肤、肺脏、乙状结肠和脾脏组织,经4%中性缓冲甲醛溶液或Bouin’s液固定后,制作常规组织切片,分别作HE、甲苯胺蓝、阿尔新蓝-藏红和P物质免疫组织化学染色,镜下观察并应用彩色病理图像分析软件进行形态学分析;另取皮肤组织固定于磷酸缓冲戊二醛溶液,制作常规超薄切片,透射电镜下观察肥大细胞超微结构。结果六种动物不同组织中肥大细胞各有其分布特点,且在形态大小、异染性及染色特性等方面表现各异,肥大细胞密度和形态学参数差异有显著性（P〈0.05）;豚鼠、犬和猴皮肤肥大细胞颗粒具有特殊亚微结构;小鼠皮肤组织内可见P物质免疫阳性肥大细胞和神经纤维,犬脾脏内可见P物质免疫阳性肥大细胞。结论在六种实验动物的同一组织中以及同一种动物不同组织中,肥大细胞具有明显的异质性,此异质性对采用实验动物开展与肥大细胞功能相关的动物实验研究具有重要参考价值。     

几种肥大细胞染色方法的比较

运用ABC—爱先蓝—PAS混合染色及PAP技术对大鼠肝、胃、肠及DAB诱发的大鼠肝癌中肥大细胞进行了染色对比实验,结果表明:Carnoy及福尔马林等固定液固定的组织内肥大细胞均能被爱先蓝染色成蓝色,其染色时间不同,该种染色方法可作为一种常规的染色方法,用于确定肥大细胞在组织中的分布及数量。ABC—爱先蓝—PAS混合染色方法及PAP技术均能准确、有效地确定肥大细胞(MC)性质。ABC—爱先蓝—PAS混合染色使结缔组织肥大细胞(CTMC)呈棕褐色,周边略蓝色。粘膜肥大细胞(MMC)则呈蓝色,通过不同颜色的显示,可清楚地将CTMC与MMC区分开来。PAP方法具更高的特异性。但有关的抗体来源缺乏,因此在无特异Ⅰ抗体的情况下,ABC—爱先蓝—PAS混合染色方法亦可视为鉴别两类不同性质MC的一种较为理想的方法。     

苦味酸—甲醛固定液

PF是两仲药品即苦味酸(Picric acid)和甲醛(Formaldehyde)的英文字头。PF固定液是依据Bouin氏液改良而来,去掉了其中的冰醋酸,配制比例也作了改动。主要用于免疫组织化学和生物组织化学的研究,它能较好地固定组织、细胞和抗原。其配方如下:     

小肠上皮分泌细胞特性的组织学观察

目的寻找一种可以替代人体消化管的动物标本,并通过特殊染色方法,使得小肠上皮分泌细胞的形态特征能够明显地显示出来。方法随机采集成年猫小肠的新鲜标本,经Bouin液灌注固定24h后,石蜡包埋切片脱蜡入水。分别采用Gomori染色法、PAS反应、Gomori＋PAS反应、阿利新蓝（alcian blue,AB）染色法、AB＋PAS反应、HE染色法和苏木精-焰红染色法进行染色。结果在各种染色的切片标本上,能够观察到杯状细胞的形态、分布和染色特性以及肠内分泌细胞的特点,并发现在它们之间还存在一种绿色颗粒细胞和嗜酸性颗粒细胞。结论通过特殊染色可以肯定猫的小肠杯状细胞合成的是中性粘蛋白和酸性粘蛋白;绿色颗粒细胞为未成熟杯状细胞;嗜酸性颗粒细胞为Paneth细胞,其特点是单个分散分布。肠内分泌细胞与周围其他上皮细胞的染色对比明显而容易识别。     

不同固定液对豚鼠免疫器官石蜡切片质量的影响

摘要 目的：比较不同固定液对SPF级豚鼠免疫器官的固定效果,为筛选适用于脾脏、淋巴结和骨髓的固定液提供参考。方法：将21只300～350 g雄性SPF级豚鼠随机分为7组,每组3只,摘取脾脏、肠系膜淋巴结、髂骨骨髓,各组分别使用4 %多聚甲醛溶液、10 %中性福尔马林溶液、Bouin''s溶液、Carnoy溶液、Davidson''s溶液、Zenker溶液、Helly溶液固定48 h。髂骨骨髓在固定前浸泡于EDTA脱钙液（pH7.2）中并置于37 ℃孵箱进行脱钙。通过制作石蜡切片并进行HE染色,从切片龟裂程度、细胞形态、染色效果等方面比较7种固定液对切片质量的影响。结果：10 %中性福尔马林溶液固定后的脾脏未出现龟裂,白髓和红髓着色清晰,淋巴细胞形态易辨。Carnoy溶液固定后的淋巴结生发中心明区暗区分明,红蓝染色适中,淋巴细胞形态清晰。4 %多聚甲醛溶液固定后的骨髓着色适中,清晰可辨。结论：常温条件下对组织固定48 h,经HE染色后想要达到理想的切片质量,建议使用10 %中性福尔马林溶液固定脾脏,使用Carnoy溶液固定淋巴结,首选4 %多聚甲醛溶液固定骨髓,如果考虑固定的同时完成脱钙建议选择Bouin''s溶液固定骨髓。     

牛蛙肥大细胞的组织化学与形态学

目的鉴定牛蛙组织中肥大细胞的存在。方法用于肥大细胞研究的一些常规组织化学技术与形态学方法。结果牛蛙的舌、肠、肠系膜和脾中肥大细胞数量较多,少量也见于神经、心、肾、肝和皮肤等多种组织中。肥大细胞有沿血管周和神经分布的倾向。脾脏中的肥大细胞形状比较一致,呈圆形或卵圆形,而在其它部位的肥大细胞则形态多样。Bouin氏液及Carnoy氏液是牛蛙肥大细胞优良的固定液。然而,与哺乳动物的黏膜肥大细胞相似的是,中性缓冲福尔马林(NBF)固定显著的阻断了牛蛙肠黏膜肥大细胞(MMC)的染色。有趣的是,甲苯胺蓝是牛蛙肥大细胞的最佳染料,它比阿尔新蓝能很好地显示牛蛙的肥大细胞。透射电镜下证实,牛蛙肥大细胞中含有大量特征性的胞浆颗粒。肥大细胞靠近雪旺氏细胞,并可见于神经束膜间,甚至以其突起与神经束膜相连。结论通过组织化学与形态学研究证实了牛蛙组织中肥大细胞的存在,再次证实肥大细胞与外周神经之间存在密切的解剖学关系。     

Periodic acid incubation can replace hydrochloric acid hydrolysis and trypsin digestion in immunogold-silver staining of bromodeoxyuridine incorporation in plastic sections and allows the PAS reaction

Summary We have examined the possibility of improving the present methods of detecting bromodeoxyuridine (BrdU) and for combining the PAS reaction with the BrdU detection by means of immunogold-silver staining (IGSS). This was done in testes fixed in Carnoy or Bouin, and in parts of the small intestine which were fixed in Carnoy or periodate-lysine-paraformaldehyde (PLP). All tissues were embedded in a mixture of glycol methacrylate and butanediol-monocrylate. It was found to be impossible to carry out BrdU detection using HCl hydrolysis and trypsin digestion in combination with a PAS reaction. However, incubation of the plastic sections in periodic acid for a period of 30 minutes appeared to make it possible to eliminate the HCl denaturation step and to carry out a specific PAS reaction. Moreover, after incubation in periodic acid, trypsin digestion was no longer required to make the BrdU label accessible in GMA-embedded sections, nor to re-expose the antigenic sites in plastic sections of tissues fixed with cross-linking fixatives. In this way the loss of cell structures, which is inevitable when trypsin is used, can be avoided. Now a BrdU detection with improved morphology can be combined with the PAS reaction in the same plastic section in order to stain tissue carbohydrates. This is important for tumour diagnosis, where the PAS reaction can be very useful.     

Application of alternative fixatives to formalin in diagnostic pathology

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.     

The effect of fixatives in the quantification of morphological lipofuscin as an age index in crustaceans

The role of fixation in studies using histological techniques to measure lipofuscin as an age indicator was evaluated. Four fixatives were used: Bouin, 10% buffered saline formaldehyde, Davidson and San Felice. The species used was the Norway lobster (Nephrops norvegicus). Similar sized males, with a maximum carapace length difference of less than 10 mm were used and attributed randomly to each treatment. Results showed that San Felice and Davidson were not appropriate for lipofuscin studies. Bouin and 10% formaldehyde produced the best results in terms of tissue quality and lipofuscin granule brightness. This work suggests that Bouin can be a substitute of formaldehyde in lipofuscin studies, in particular in situations when other structures, such as gonads, are needed for parallel or complementary studies.     

APPSWE转基因小鼠发育过程中海马CA1区神经细胞凋亡研究

目的探讨APPSWE转基因小鼠发育过程中海马CA1区神经细胞凋亡规律。方法取不同发育时间（P0、P7、P14、P30、P00、P180）APPSWE转基因模型鼠与同时问点对照鼠,Nissl染色观察海马结构和锥体细胞形态,免疫组织化学方法观察海马细胞内Caspase-3表达变化,RT—PCR检测Caspase-3 mRNA表达变化。结果随着小鼠的生长发育,P14时间点以后,模型组CA1区神经元Caspase-3阳性细胞密度比对照组高,RT—PCR检测结果与Caspase-3免疫组织化学结果基本一致。结论APPSWE转基因小鼠发育中的海马神经细胞过度凋亡可能与阿茨海默病的发生、发展具有联系。     

Alcoholic fixation over formalin fixation: A new,safer option for morphologic and molecular analysis of tissues

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher (P < 0.05) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.     

Staining methods for Calcium—a Comparison Based on Reactions Shown by Cardiac Calcinosis in DBA Mice

Spontaneous calcification, which occurs frequently in hearts of mice of the DBA strain, has been used to compare effectiveness of calcium stains. Three fixatives (1) 80% ethanol, (2) 10% formalin and 3.5% acetic acid in 95% ethanol (modified Lavdowsky's) and (3) a 1:1 mixture of concentrated formalin and absolute ethanol were tested for their subsequent effects on staining. Stains for Ca included purpurin, von Kossa's, phthalocyanin, and glyoxal bis(2-hydroxyanil). These, with appropriate counterstaining, were compared among each other, with hematoxylin-eosin (H-E), and with the periodic acid-Schiff (PAS) reaction. Fixatives (1) and (3) did not affect results appreciably, but the von Kossa technic was unsatisfactory after (2). All stain demonstrated sites of Ca deposits, but purpurin and von Kossa's gave greatest sharpness of definition and were the simplest to perform. H-E and PAS with Alcian blue showed the same Ca sites in addition to details of the surrounding tissue.     

时滞Nicholson＇s Blowflies方程中行波解的Hopf分支

主要利用时滞微分方程中Hopf分支理论探讨时滞Nicholson＇s Blowflies方程中行波解随时滞量τ大小变化的分支行为.结果发现时滞量经过某一数值τ_0=1/（cω_0） arcsin-cω_0/p时,原系统会产生分支现象,最终导致形成周期性行波解.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Commercial Hair Sprays as Fixatives for Hematological Cytochemistry

Commercial hair sprays have been found to be excellent cytological fixatives for a variety of enzymatic and nonenzymatic hematological staining procedures. Of the varieties evaluated, not all were found suitable for each staining procedure tested. With some preparations, excellent leukocyte morphology and preservation of reaction product was obtained after staining for carbohydrates (PAS), lipid (Sudan black), nucleic acids (methyl green-pyronin), peroxidase, M-nadi oxidase and alkaline phosphatase. These spray preparations are remarkably inexpensive, readily stored, and stable and simple to use. The fixative ability is probably related to their polyvinylpyrrolidine and alcohol content.     

Histological structure of the male reproductive organs and spermatogenesis in a copulating sculpin, <Emphasis Type="Italic">Radulinopsis taranetzi</Emphasis> (Scorpaeniformes: Cottidae)

Characteristics of the structure and function of male reproductive organs in the copulating sculpin Radulinopsis taranetzi were investigated based on histological observations. The male reproductive organs comprised three parts: a pair of testes, a seminal vesicle, and a penis. Germinal cells matured in cysts located in the small seminal lobules. Asynchronous spermatogenesis advanced rapidly from the posterior to the anterior region of the testes. After sperm matured in the posterior part of the testes, the seminiferous epithelium of the seminal lobules synthesized and secreted eosinophilic fluid that showed a positive periodic acid–Schiff (PAS) reaction into the seminal lobules. Spermatozoa excreted from the posterior part of the testes were stored together with the secretion in the seminal vesicle and showed no activity in the seminal fluid. Histological observations throughout the year suggest that the fluid is secreted and spermatozoa are stored in the seminal vesicles during February to July, which is presumably when mating occurs. The importance of testicular maturation and the secretion of eosinophilic fluid during this long reproductive period is also discussed.