Tissue preparation. Cecum samples were removed and flushed out by using saline. Samples were fixed intact in Carnoy fixative for 30 min prior to storage in 70% ethanol. Tissues were prepared by using the gut bundle technique (40). Tissues were then paraffin embedded by using standard histological techniques. Two nonserial 3-µm sections were mounted per slide and stained with hematoxylin and eosin.
Detection of apoptotic cells. Sections were stained with hematoxylin and eosin to allow the visualization of apoptotic cells. Such cells are detected on the basis of their morphology by using light microscopy, a method that has been used extensively (28, 29, 35, 41). Typically, apoptotic cells appear pink and circular, with a crescent-shaped nucleus, and are bubbled up out of the plain of focus. Tunnel labeling is another method that has can be used to detect apoptosis in the intestinal epithelium. However, this technique is prone to false-positive and false-negative results compared to morphological assessment, as well as failing to distinguish between DNA cleaved by apoptosis and DNA fragments cleaved by other processes (39). For the purpose of this investigation, therefore, morphological analysis was deemed the most reliable method to use.
Levels of epithelial cell proliferation. Groups of four mice were injected intraperitoneally with 10 mg of bromodeoxyuridine (BrdU; Sigma, Poole, United Kingdom) 40 min prior to sacrifice. All animals were killed at the same time within and between experiments to minimize any differences in proliferation attributable to variation in circadian rhythm. Detection of nuclei that had incorporated BrdU was performed by immunohistochemistry using a monoclonal anti-BrdU antibody (Mas 250b; Harlan Serum Laboratories, Loughborough, United Kingdom) as described previously (13). Sections were analyzed by scoring 50 cecal crypts per mouse with four mice per group.
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組織準備。盲腸樣品由使用去除了和驅趕了出來鹽。樣品原封被固定了在Carnoy 固定劑30 分鐘在存貯之前在70% 對氨基苯甲酸二裡。組織由使用準備了食道捆綁技術(40) 。組織是然後石蠟由使用埋置標準組織學技術。二個非系列的3-.m 部分登上了每幻燈片和被弄髒了與hematoxylin 和四溴熒光素。
apoptotic 細胞的偵查。部分被弄髒與hematoxylin 和四溴熒光素允許apoptotic 細胞的形象化。這樣細胞被查出根據他們的形態學由使用光學顯微學, 廣泛地被使用了的方法(28, 29, 35, 41) 。典型地, apoptotic 細胞出現桃紅色和圓, 以一個月牙形的中堅力量, 和起泡在焦點外面平原。隧道標記是有可能被使用查出apoptosis 在小腸皮膜的其它方法。但是, 這個技術以及不是有傾向對假正面和假消極結果與形態評估比較, 區別在DNA 由apoptosis 劈開和DNA 片段之間由其它過程(39) 劈開。為這次調查的目的, 因此, 形態分析被視為可靠方法使用。
上皮細胞擴散的水平。小組四隻老鼠腹膜內被注射了與10 bromodeoxyuridine (BrdU 毫克; 斯格碼, Poole, 英國) 40 分鐘在犧牲之前。所有動物同時被殺害在實驗之間使在擴散上的所有區別減到最小可歸屬對在合併了BrdU 由immunohistochemistry 執行使用monoclonal anti-BrdU 抗體中堅力量的晝夜生理節律偵查上變化(Mas 250b; Harlan 清液實驗室, Loughborough, 英國) 如所描述早先(13) 。部分被計分分析了50 盲腸的土窖每老鼠與四隻老鼠每小組。參考資料： 自己