细胞免疫荧光实验步骤.docx

第一天：

1. 在培养板中将已爬好细胞的玻片用PBS浸洗3次,每次3min；

2. 用4%的多聚甲醛固定爬片15min, PBS浸洗玻片3次,每次3min；

3. 0.5%Triton X-100( PBS配制 )室温通透20min（细胞膜上表达的抗原省略此步骤）；

4. PBS浸洗玻片3次,每次3 min,吸水纸吸干PBS,在玻片上滴加正常山羊血清,室温封闭30min；

5. 吸水纸吸掉封闭液,不洗,每张玻片滴加足够量的稀释好的一抗并放入湿盒,4℃孵育过夜；

第二天：

6. 加荧光二抗：PBST 浸洗爬片3次,每次3min,吸水纸吸干爬片上多余液体后滴加稀释好的荧光二抗,湿盒中20-37℃孵育1h,PBST浸洗切片3次,每次3min；注意：从加荧光二抗起,后面所有操作步骤都尽量在较暗处进行。

7. 复染核：滴加DAPI避光孵育5min,对标本进行染核,PBST 5min×4次洗去多余的DAPI；8. 用吸水纸吸干爬片上的液体,用含抗荧光淬灭剂的封片液封片,然后在荧光显微镜下观察采集图像。

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 for IF

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 SlowF ade Gold, S36937)

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