Re: [求救] IHC 不太順利

看板Biotech作者YuDavid (討厭失眠)時間17年前 (2009/03/31 20:59)推噓1(1推 0噓 5→)

留言6則, 2人參與討論串3/6 (看更多)

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

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