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）5.0.5Triton X100（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 PBS5) (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 PBS9) 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 PBS11)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)