贴壁细胞的免疫荧光染色方法.docVIP

贴壁细胞的免疫荧光染色方法 Materials: 1. PBS solution 2. 4% Paraformaldehyde (PFA fixative): Dissolve 4g paraformaldehyde in 100ml PBS solution, stir at 70℃ to dissolve; 3. PBS-T solution: (0.1% Triton X-100 in PBS solution) 4. PBS-B blocking solution: (4% BSA in PBS solution) 5. Primary antibody: Dilute with PBS-B solution, dilution factors should refer to manual, or, test from 1:50~200, should be more concentrate than application in Western blot; 6. Secondary antibody: Dilute with PBS-B solution, dilution factors should refer to manual Procedure: 1. Cultured cells, let it attach to the coverslips in 6-well plate; 2. Remove medium, rinse with PBS twice; 3. Add 2ml 4% paraformaldehyde, incubate at room temperature for 20 minutes; 4. Rinse with 2ml PBS three times, rinse for 5 minutes every time; 5. Permeabilize cells with 2ml PBS-T solution at 4?C for 10 minutes; 6. Remove PBS-T solution, rinse cells with PBS for 5 minutes at room temperature; 7. Block non-specific interaction with PBS-B solution at 37?C for 30 minutes; 8. Add primary antibody solution, incubate at 4?C overnight; 9. Remove primary antibody solution, wash with PBS for 5 minutes; 10. Add secondary antibody solution, incubate at room temperature for 1 hour; 11. Wash with PBS three times, 5 minutes each; 12. Add anti-fade DAPI solution if needed; 13. Observation. 细胞免疫荧光步骤 1.细胞爬片； 2.4%多聚甲醛固定10min(固定细胞器用预冷的70%甲醇+30%丙酮)； 3.PBS漂洗5min； 4.0.5% Triton 穿孔15min(丙酮固定法不用透化处理)； 5.PBS漂洗2次，每次5min； 6.1%BSA封闭30min； 7.加入1%BSA稀释的一抗，于37℃杂交2h； 8.PBS漂洗2次，每次5min； 9.加入1%BSA稀释的二抗，于37℃杂交1h； 10.PBS漂洗2次，每次5min； 11.5ug/ml DAPI染色2min； 12.抗淬灭封片剂封片。 抗淬灭封片剂: 2.5% DABCO (w/v) 50mM Tris(pH8.0) 90%甘油 作为荧光显微镜使用： （1）先关闭荧光通道，再打开荧光激发器电源，等待15分钟。 （2）等待期间，打开显微镜光源，找到需要观察的样品，选用合适的物镜，调整焦距。 （3）关闭显微镜光源，打开荧光通道。 （4）需要图片，要把镜体上的相应拉杆拉到绿线位子。按下相应附件上的照相按扭。 作为荧光显微镜使用： （1）先关闭荧光通道，再打开荧光激发器电源，等待15分钟。 （2）等待期间，打开显微镜光源，找到需要观察的样品，选用合适的物镜，调整焦距。 （3）关闭显微镜光源，打开荧光通道。 （4）需要图片，要把镜体上的相应拉杆拉到绿线位子。按下相应附件上的照相按扭。 细胞免疫荧光 细胞爬片 4%多聚甲醛固定10min (固定细胞器用预冷的70%甲醇+30%丙酮) PBS漂洗5min 0.5% Triton 穿孔15min