免疫荧光protocol.doc

细胞免疫荧光步骤
1.在24孔板里加500微升培养基，放爬片，接种细胞（做实验以30-50%汇合度较好。10000-30000左右
2.给药处理24h。
3.PBS洗三遍。
4. 4％冷的多聚甲醛固定15分钟，PBS洗三遍，每次5min，摇床。（避光）
5.0.5％Triton X－100（PBS配）破膜15min，PBS洗三遍，每次5min，摇床。
6.5%BSA（牛血清白蛋白，PBS配）封闭60分钟,不用洗。
7.加一抗孵育（5%BSA配），4℃摇床过夜。
8. 收集一抗，PBS洗三遍，每次5min，摇床。孵育二抗Alexa Fluor 488（1:1000 ）,室温60min（避光）
9.回收二抗，PBS洗三遍，摇床，每次5min。
10.0.5ug/mLDAPI（5%BSA配，2滴/ml）染核15min。（避光）
11. PBS洗三遍，每次5min，摇床。
12.取载玻片，滴加10uL抗荧光衰减封片剂，将爬片有细胞面盖在封片剂上，指甲油封片子的对角线。
All steps usually at RT.
1)    Remove culture medium and fix cells (a common fixative is 4% formaldehyde in PBS, for 15 minutes)
2)    Wash well in PBS (3 x 5 minutes is typical)
3)    Permeabilize the cells (a common permeabilization reagent is 0.2% Triton X-100 in PBS for 30 minutes)
4)    Wash well in PBS
5)    (optional:  Block for non-specific dye binding using the Image-iT FX Image Enhancer Solution, I36933)
6)    Block for non-specific antibody binding 30-60 minutes (a common blocking solution would be 3-6% bovine serum albumin / 5% normal goat serum / PBS, or commercial blocking reagents like our BlockAid, product B10710)
7)    Incubate in primary antibody for 30-60 minutes, in blocking solution or overnight at 4 degrees (antibody concentrations vary, but usually between 0.5-10ug/mL)
8)    Wash well in PBS
9)    Incubate in secondary antibody for 30-60 minutes, in 3-6% bovine serum albumin / PBS  (a good starting antibody concentration is 5 ug/mL)
10) Wash well in PBS
11) Counterstain as needed (such as with DAPI, D1306)
12) Mount in appropriate mounting medium (for fluorescent secondaries, a good antifade solution is best, such as ProLong Gold, P36934, or SlowFade Gold, S36937)