免疫荧光protocol

更新时间:2023-08-11 05:09:55 阅读: 评论:0

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.性感的英文5%Triton X-100PBS武汉新东方学校配)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,摇床孵育英语四级多少分算过二抗Alexa Fluor 488(1:1000 ),室温60min(避光)
9.回收二抗,PBS洗三遍,摇床,每次5min
attach是什么意思
10.0.5ug/mLDAPI两颊毛孔粗大怎么办(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)    pope francisPermeabilize the cells (a common permeabilization reagent is 0.2% Triton X-100 in PBS for 30 minutes)
经济学人中文网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 rum albumin / 5% normal goat rum / PBS, or commercial blocking reagents like our BlockAid, product B10710)
里外里
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 condary antibody for 30-60 minutes, in 3-6% bovine rum albumin / PBS  (a good starting antibody concentration is 5 ug/mL)
curitykiss10) Wash well in PBS
11)charter Counterstain as needed (such as with DAPI, D1306)
12) Mount in appropriate mounting medium (for fluorescent condaries, a good antifade solution is best, such as ProLong Gold, P36934, or SlowFade Gold, S36937)
 

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标签:细胞   片剂   荧光   微升   毛孔   片子   孔板
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