作者YuDavid (討厭失眠)
看板Biotech
標題Re: [求救] IHC 不太順利
時間Tue Mar 31 20:59:27 2009
筆記本放在實驗室 所以protocol 可能書寫不是很正規...請見諒
1. Deparaffinze sections and rehydrate as follows
xylene for 5'
100% ethanol for 3'
95% ethanol for 3'
80%~~
70%~~
55%~~(依此類推@@)
wash with 1X PBS for 3'
2.Blocking endogenous peroxidase
Soak slides in 0.3% hydrogen peroxide(diluted with PBS) for 8~10 min at RT
wash with 1X PBS for 3'
3. Antigen retrieval
Soak slodes in bioled citrite buffer saline for 10~25 min
wash with 1X PBS for 3'
4. Blocking with goat serum for 15~20 min
wash with 1X PBS for 3'
5% serum is diluted with PBS
5. 1st antibody for 1 hr at RT
wash sections with 1X PBS for 3'
6. 2nd Ab for 1hr at RT
Abs are diluted 1:100 with serum blocking buffer(5% goat serum)
wash in PBS 3'
7. Detection performed by DAB
DAB:PBS=1:20 in the dark for 8~10 min(我不懂為甚麼要避光 但是學姊說要)
wash in PBS for 3'
8. Counterstain with hematoxylin for 3'
wash in PBS or ddH2O
9. Dehytrate sections
50% ethanol 3'
70%~~
80%~~
95%~~
100%~~
Xylene 5'
10. Covering slide
也有些人先做retrieval再做 endogenous peroxidase blocking
但我認為這兩個步驟的先後似乎沒差吧?
至於一抗 我甚至看過有人放RT overnight的 這樣不是很容易造成"假陽性"嗎
由於sample可能會泡太久脫落 所以不敢放overnight
autoclave法做retrieval步知道需不需要泡在buffer裡面呢?請高手指點
我是沒有泡啦 一樣是怕脫落 囧
總之是新手 完全是自己摸索
希望能聽到各位強者分享經驗並且指教 感激不盡
--
※ 發信站: 批踢踢實業坊(ptt.cc)
◆ From: 115.192.189.245
1F：→ allisa:retrieval一定要泡在buffer裡 或你試看看用trypsin做 04/01 02:29
2F：→ allisa:一抗的話 泡久不會使sample更容易掉 不過wash要小心就是了 04/01 02:30
3F：→ allisa:一抗要放多久要試過condition才知 我用過要over 2 night的 04/01 02:31
4F：→ allisa:DAB不用避光 不過我也看過別家的protocol要避 我也不知為何 04/01 02:32
5F：推 loneangle:DAB會受到光的影響 會變弱的樣子 所以要避光 04/08 10:35
6F：→ loneangle:原液也要避光 用久了顏色變深 作用時間就要拉長 04/08 10:36