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即将举行的网络研讨会:实现无需离心的流式细胞术样品制备工作流程的标准化

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新闻 免疫荧光染色的介绍
免疫荧光染色的介绍

 免疫细胞化学基于抗原抗体反应的原理。免疫荧光染色首先用荧光素标记已知的抗原或抗体,制成荧光标记物,然后用荧光抗体(或抗原)作为分子探针,检查细胞或组织中相应的抗原(或抗体)。在细胞或组织中形成的抗原-抗体复合物含有荧光素。用荧光显微镜观察标本时,荧光素在激发光的照射下发出明亮的荧光(黄绿色或橙红色)。免疫荧光染色可以看到荧光所在的细胞或组织,从而确定抗原或抗体的性质和位置,通过定量技术确定含量。免疫荧光也叫免疫荧光染色。荧光实验的原理是将荧光素标记在相应的抗体上,直接与相应的抗原反应。其优点是方法简单,特异性高,非特异性荧光染色少,标记抗体用量相对较大。抗原-抗体反应用于定位组织或细胞中的抗原物质。

  下面详细描述免疫荧光染色的步骤:

  1.将0.01mol/L ph 7.4的PBS滴在待测样品上,10分钟后弃去,使样品保持一定的湿度。

  2.滴加适当稀释的荧光标记抗体溶液至完全覆盖标本,置于有盖搪瓷盒中,保温一定时间(参考:30min)。

  3.取出载玻片,放在载玻片架上,用pH7.4的0.01mol/L PBS冲洗,然后依次浸泡在三缸pH7.4的0.01mol/L PBS中,每缸持续3-5 min,不时摇动。

  4.取出载玻片,用滤纸吸干多余的水,但不要使标本变干,加入一滴缓冲甘油并用盖玻片盖住。

  5.免疫荧光染色立即用荧光显微镜观察。观察标本的特定荧光强度,一般可用“+”表示:(-)无荧光;(正负)微弱的可疑荧光;(+)荧光微弱,但清晰可见;(++)明亮的荧光;(++++-++)荧光且有光泽。当待检标本的特异性荧光染色强度达到“++”或以上,各种对照显示(+)或(-),即可判定为阳性。


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