细胞免疫荧光实验步骤.docxVIP

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%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?moun