Immunofluorescent Protocol
Hearts were fixed in 4% paraformaldehyde overnight at room temperature. The following day, hearts were moved to 50% ethanol and stored for up to one week before paraffin embedding. Paraffin ctions (5 μm) were cut through the entire ventricle. All immunofluorescence was performed on paraffin ctions. For phosphohistone H3/troponin T and Wt1/Troponin T co-staining, slides were rind 3 times in PBS, blocked in 10% goat rum for 20 minutes followed by 3 rins in PBS. Sections underwent antigen retrieval by boiling in sodium citrate solution for 20 minutes. This was followed by overnight incubation with primary antibodies against phospho-histone H3 (Ser10) (1:100, rabbit polyclonal, Millipore, MA) or Wt1 (1:50, rabbit polyclonal, Santa Cruz Biotechnology, CA) and cardiac troponin T (1:100, mou monoclonal, Thermo Scientific, IL). The following day, slides were washed 3 times in PBS and incubated with anti-mou and anti-rabbit condary antibodies conjugated to FITC or TRITC for 1 hour at room temperature. Slides were washed 3 times in PBS, stained with Hoechst 33342 or DAPI for 3 minutes to label nuclei and mounted in Vectashield.
洛阳好玩的地方
1.The protocols are from method and material of “Enzo R. Porrdllo, Ahmed I. Mahmoud, Emma Simpson, et al. Transient regenerative potential of the neonatal mou Heart[J]. Science, 2011, 331(6020): 1078-108.”
2.Harvard H&E staining Protocol
免疫荧光三标实验方案
Ab1与某种细胞表面抗原结合
古代神话Ab2与某种细胞骨架
DAPI标记细胞核
一、脱蜡至水
1. 石蜡切片56℃ 预热3h。
2. 二甲苯Ⅰ、二甲苯Ⅱ、二甲苯Ⅲ 各3min脱蜡
梭子蟹好吃吗
3. 无水乙醇Ⅰ和无水乙醇Ⅱ复水3min。
4. 95%、90%、80%、70%的乙醇各3min。
5. 去离子水洗2×3min。
二、抗原修复
6. 切片置0.01M PH6.0 柠檬酸盐缓冲液中再放入高压锅，盖上盖子及高压阀，待有蒸汽冒出开始计时加热15min。（或CB液中微波中高火5min煮沸，然后中火维持沸腾20min）
7. 将高压锅置流水下冲洗减压，安全打开高压锅。(微波修复无此步骤）
8. 让载片在原柠檬酸盐缓冲液中自然冷却至室温，降至室温前切勿取出。
9. 自来水下缓流冲洗3min。
耶律10.去离子水洗3min×1次。
11. PBS洗3min×2次。
中学生书包三、免疫标记
12. 甩干PBS，正常山羊血清封闭，37℃，45min。首页不显示页眉
13. 甩去山羊血清，勿洗，种属来源不同的Ab1和Ab2按适当比例混合后滴加于切片样品位置，4℃过夜（或37℃ 1h）。注意设置对照.（对照可分为阴性对照、阳性对照和空白对照，根据实验需要设置）。
14. 室温复温45min。
15. PBS洗5min×3次。
16. 甩去PBS，滴加相应的适当浓度的二抗及DAPI混合物（我这里使用FITC标记的二抗与Ab1结合，TRITC标记的二抗与细胞骨架蛋白结合，DAPI是细胞核DNA荧光染料）。37℃处理1h。
17. PBS洗5min×4次。家长评语大全
18. 甘油封片，荧光显微镜观察或显微照相。
注意：在实验过程中勿使切片样品变干，以免影响实验效果
手写文字大鼠造血干细胞免疫荧光三标图片（图中核标为蓝色，细胞质标记为红色，细胞膜标记为绿色，红绿共标记区为黄色，红、绿、蓝共标记区为白色）。
