贴壁细胞的免疫荧光染色方法Materials:1. PBS solution2. 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 manualProcedure: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5min0.5% Triton 穿孔15min(丙酮固定法不用透化处理)PBS漂洗2次，每次5min1%BSA封闭30min加入1%BSA稀释的一抗，于37℃杂交2hPBS漂洗2次，每次5min加入1%BSA稀释的二抗，于37℃杂交1hPBS漂洗2次，每次5min5ug/ml DAPI染色2min抗淬灭封片剂封片2.5% DABCO (w/v)抗淬灭封片剂50mM Tris(pH8.0)90%甘油0.25g DABCO9ml甘油0.5ml 1M Tris(Ph8.0) 70℃溶解，-20℃保存0.5ml ddH2O2005-07-25 20:25免疫荧光第一步的步骤几注意事项1.首先是玻片的处理:泡酸去污, 冲洗, 晾干, 泡纯酒精脱脂, 蒸馏水洗(此时要用镊子夹着洗), 晾干备用.2.不知道你做的细胞是贴壁比较好的细胞比如干细胞,还是贴壁不大好的细胞比如神经元前者的话可以不用多聚赖氨酸包被玻片,后者爬片之前要包被多聚赖氨酸以免洗片时细胞掉片3. 固定,我用的是预冷纯丙酮固定15分钟(应为丙酮有毒,操作的时候小心些),这步应该注意:丙酮很容易挥发,但又要保证固定期间不能干片,所以要多滴加丙酮,其间用盖子盖住,这一步不要在6孔板内进行,因为丙酮会将其熔化.4. 不知道你的一抗是在胞浆表达还是在胞核表达如果在胞浆表达,可以不用0.01%的TRITON X-100渗透(我前几次用了TRITON,结果胞核也表现出很强的荧光), 当然,如果一抗是在胞核内表达要用TRITON 渗透15分钟5. 5%的正常山羊血清或是5%的BSA封闭,注意这一步不要洗,只是甩掉多余的封闭液.6. 加一抗,根据你所做抗体的需求稀释,一般是先将稀释液加到EP管里,后加一抗.(一抗在吸出来之前要离心10-20秒),一抗的量要多加些,也是为了避免干片.7. 孵育抗体:最好是4度冰箱过夜,这个过夜不是简单的过夜,一般要达14-16个小时,此时要将6孔板做成湿盒,目的是妨干片.在孵育期间不要随意搬动,以免抗体流失.注意,PBS液的PH值要调在7.4左右孵育一抗不能干片,孵育一抗时间要久;其四,多余的封闭液不能洗掉,只能甩干2005-07-25 20:51总之,首先,应该注意的是片子有没进行了脱脂处理;其二,注意洗片用PBS的PH植要在7.4左右;其三,固定,孵育一抗不能干片,孵育一抗时间要久;其四,多余的封闭液不能洗掉,只能甩干;其五,要注意避光说了这么多,嘿嘿,最重要的一点是:你以后阐述问题的时候请具体一点,不要让回答问题的人遍地撒网,好累呀2009-01-23 10:50首先用16孔板没有任何问题分析没有荧光的结果1. 固定最好用4%多聚甲醛30min2. 一抗孵育时间太短我的经验37度2小时或者4度过夜效果更好3. 二抗孵育时间是多少？建议37度2--3小时我也有做过5个小时的最后你用的是那种ccd的荧光显微镜吗？有的时候染的不好镜下看不到荧光但是是可以照出来的！！Fix cells in 2% formaldehyde in PBS/pH 7.4 for 15 min. at 20oC. 2% formaldehyde is made up fresh prior to use by dissolving the appropriate amount of EM grade paraformaldehyde (Prill form, from Electron Microscopy Sciences) in PBS and heating on a hot plate in the hood (setting of 5-6) until the aldehyde goes into solution. Keep the bottle cap loosened so that pressure does not build up. Cool down to 20oC and pH to 7.4. Alternatively, cells may be fixed in 100% methanol at -20oC for 3 minutes. If methanol fixation is used skip to step 4.Wash in PBS 3 X 10 min.Permeabilize in 0.2% Triton X-100 plus 1% normal goat serum (NGS) in PBS/pH 7.3 for 5 minutes on ice.Wash in PBS + 1% NGS 3 X 10 min.Incubate in the appropriate concentration of primary antibody for 1 hour at room temp. in a humidified chamber. If using 22mm X 22mm square coverslips, 30 ul of diluted antibody is placed on the coverslip and the coverslip is inverted onto a glass slide. The slide is then placed in the humidified chamber which is incubated at room temp.Wash in PBS + 1% NGS 3 X 10 min.Incubate in secondary antibody (FITC or Texas Red conjugated) at a dilution of 1:50 for 1 hour in a humidified chamber at room temp.Wash in PBS 4 X 10 min.Mount coverslip with a drop of mounting medium (see Fluorescence Mounting Medium Protocol) and seal coverslip with clear nail polish to prevent drying and movement under the microscope.liguofan wrote:多聚甲醛固定[24孔中加多少啊？],0.5-1.0 ml;liguofan wrote:加1抗，[不用湿盒，如何防干燥？用个大饭盒，加上水，盖上盖，过夜？]parafilm覆盖，或将coverslips倒扣在载玻片上后照你的方法，另外饭盒里可以放上纱布；liguofan wrote:封片[如何做？]如果立即观察、拍照，可以用PBS封片，若放置一定时间则请用水溶性封固剂封片并置4度保存。