免疫荧光protocol

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。

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)