信帆生物为您提供:石蜡切片免疫荧光实验步骤
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信帆生物为您提供:石蜡切片免疫荧光实验步骤

 1、 石蜡切片脱蜡至水:依次将切片放入二甲苯15min-二甲苯15min-无水乙醇5min-无水乙醇5min-85%酒精5min-75%酒精5min-蒸馏水洗。

2、 抗原修复:组织切片置于盛满EDTA抗原修复缓冲液PH9.0的修复盒中于微波炉内进行抗原修复。中火至沸后断电间隔10min中低火至沸,此过程中应防止缓冲液过度蒸发,切勿干片。自然冷却后将玻片置于PBSPH7.4中在脱色摇床上晃动洗涤3次,每次5min(修复液和修复条件根据组织来确定)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      

3、 BSA封闭:切片稍甩干后用组化笔在组织周围画圈(防止抗体流走),在圈内滴加3%BSA均匀覆盖组织,室温封闭30min

4、 加一抗:轻轻甩掉封闭液,在切片上滴加PBS按一定比例配好的一抗,切片平放于湿盒内4°C孵育过夜。(湿盒内加少量水防止抗体蒸发)

5、 加二抗:玻片置于PBSPH7.4中在脱色摇床上晃动洗涤3次,每次5min。切片稍甩干后在圈内滴加与一抗相应种属的二抗覆盖组织,避光室温孵育50min

6、 DAPI复染细胞核:玻片置于PBSPH7.4中在脱色摇床上晃动洗涤3次,每次5min。切片稍甩干后在圈内滴加DAPI染液,避光室温孵育10min

7、 封片:玻片置于PBSPH7.4中在脱色摇床上晃动洗涤3次,每次5min。切片稍甩干后用抗荧光淬灭封片剂封片。

8、 镜检拍照:切片于尼康倒置荧光显微镜下观察并采集图像。(紫外激发波长330-380nm,发射波长420nmFITC绿光激发波长465-495nm,发射波长515-555 nmCY3红光激发波长510-560,发射波长590nm

信帆生物为您提供:石蜡切片免疫荧光实验步骤

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