精心整理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） ；4.PBS 浸洗玻片 3 次，每次 3min，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， 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精心整理左右 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 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)
