细胞免疫荧光实验步骤

细胞爬片免疫荧光实验步骤

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

7.复染核：滴加DAPI避光孵育5min，对标本进行染核，PBST5min×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。?

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?forIF

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?Enha ncer?Solution,?I36933)?

6)????Block?for?non-specific?antibody?binding?30-60?minutes?(a?common?blocking?soluti on?would?be?3-6%?bovine?serum?albumin?/?5%?normal?goat?serum?/?PBS,?or?commercial?blo cking?reagents?like?our?BlockAid,?product?B10710)?

7)????Incubate?in?primary?antibody?for?30-60?minutes,?in?blocking?solution?or?overnig ht?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?