[求救] Immunofluorescence
各位朋友～
最近在做Immunofluorescence 一直出現有nonspecific....
不知道如何解決他＠＠ 困擾我好幾個月
我要染的protein（一種cytokines）會在細胞質出現
我有作一組negative control 一直在核中出現nonspecific
不知道要怎麼解決
以下是我的實驗步驟：
1. Add 3% paraformaldehyde at RT for 15 min
2. Wash 2 times with PBS, 10 min each
3. Add 0. 1% Triton X-100 (diluted with PBS) at RT for 10 min
4. Rinse 3 times with PBS, 5 min each
5. Add blocking buffer (0.5% BSA or Normal Rabbit Serum in PBS) 30 min, 37℃
6. Add 1st Ab (Rabbit anti-CCL28) at O/N, 4℃
7. Wash 2 times with PBS, 10 min each
8. Add 2nd Ab (Goat Anti-Rabbit) for 1 hr, RT
9. Wash 2 times with PBS, 10 min each
10. Counterstain: DAPI (0.1 ug/ml in PBS) 30 sec, RT
11. Wash 2 times with PBS, 10 min each
請問一下我的步驟有哪邊是錯誤的或需要修改的
ps.我也有試過IHC，一樣也是核會有nonspecific....
謝謝大家指教了
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