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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 sections (5 μm) were cut through the entire ventricle. All immunofluorescence was performed on paraffin sections. For phosphohistone H3/troponin T and Wt1/Troponin T co-staining, slides were rinsed 3 times in PBS, blocked in 10% goat serum for 20 minutes followed by 3 rinses 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, mouse monoclonal, Thermo Scientific, IL). The following day, slides were washed 3 times in PBS and incubated with anti-mouse and anti-rabbit secondary 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.
1.These protocols are from method and material of “Enzo R. Porrdllo, Ahmed I. Mahmoud, Emma Simpson, et al. Transient regenerative potential of the neonatal mouse Heart[J]. Science, 2011, 331(6020): 1078-108.”
2.Harvard H&E staining Protocol
免疫荧光三标实验方案
免疫实验荧光器材
避光场所,湿盒,烘箱等加温设备,吸水纸,移液枪,1.5ml EP管,微波炉,抗原修复用的容器,ph试纸或ph仪,盖玻片,多聚赖氨酸(或Apes)包被的载玻片等。
免疫荧光实验试剂
Ab1与某种细胞表面抗原结合、Ab2与某种细胞骨架
相应荧光二抗
Hoechst 33342或DAPI(与双链DNA结合,显核,蓝荧光)
抗体稀释液(含1%BSA的PBS)
10×PBS
NaCl 85g, NaH2PO4·2H2O 2.964g, Na2HPO4·12H2O 19g, 定容至1000ml,配完最好高压灭菌一下)。
1×PBS
10×PBS稀释10倍,再调PH至7.2-7.4。
10×柠檬酸盐抗原修复液
(C6H8O7·H2O 3.99g, Na3C6H5O7·2H2O 23.8g,定容至1000ml,配完最好高压灭菌一下)。
1×柠檬酸盐抗原修复液
10×柠檬酸盐抗原修复液稀释10倍,再调ph至6.0。
封片剂
0.672g NaHCO3 加40ml蒸馏水,以1mol/L的NaOH调其ph至9.0左右。吸取1ml此液体,与1ml 100%甘油混匀即可。
蒸馏水
实验程序
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5min脱蜡
3. 无水乙醇Ⅰ和无水乙醇Ⅱ复水2min。
4. 95%、90%、80%、70%的乙醇各2min。
5. 去离子水洗3min。
6. 1×PBS洗3min×2次。
二、抗原修复
7. 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),将高压锅置流水下冲洗减压,安全打开高压锅。
7’. 将切片置于0.01mol/L且ph=6.0的枸橼酸液修复液中,用微波炉中火加热6分钟至微微沸腾,再用中低火力维持10分钟,停止加热后自然冷却至室温。(煮沸6分钟×4次可能更好)。
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. PBS浸洗2分钟后滴加0.3%tritonX-100透膜剂透膜12分钟(不透膜也没关系,反正细胞早就被切开了)。但注意细胞膜表面的整合蛋白不能加tritonX-100,否则会被洗掉。
10. PBS洗3min×2次。
三、免疫标记
11. 甩干PBS,正常山羊血清在湿盒中37℃封闭30分钟。
12. 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,湿盒中4℃过夜(或37℃ 2h)。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)。
13. 室温复温45min。
14. PBS洗3min×5次。
15. 甩去PBS,滴加相应的适当浓度的二抗(我这里使用FITC标记的二抗与Ab1结合,TRITC标记的二抗与Ab2结合)。37℃湿盒中孵育1h。
16. PBS洗5min×3次。
17. 滴加 Hoechst 33342(或DAPI)避光孵育2分钟,可对标本进行显核,蓝色荧光,效果极佳。
18. PBS洗2min×4次
18. 封片剂封片,荧光显微镜观察或显微照相。
注意:
1. 在实验过程中勿使切片样品变干,以免影响实验效果。
2. 若一张切片同时做两种抗体的免疫荧光,则加一抗时可将两种一抗按各自所需的浓度混合,但要注意抗体要来源于不同的动物。加二抗时也可将两种二抗按各自所需的浓度混合,但要注意二抗要有不同的颜色。
3. 第7步和第7'步任选一。
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