Hoechst 33342,也称bisBenzimide H 33342或HOE 33342,是一种可以穿透细胞膜的蓝色荧光染料,对细胞的毒性较低。Hoechst 33342染色常用于细胞凋亡检测,染色后用荧光显微镜观察或流式细胞仪检测。Hoechst 33342也常用于普通的细胞核染色,或常规的DNA染色。Hoechst 33342的最大激发波长为346nm,最大发射波长为460nm;Hoechst 33342和双链DNA结合后,最大激发波长为350nm,最大发射波长为461nm。本Hoechst 33342染色液为即用型,可直接用于固定细胞或组织的细胞核染色,也可直接用于活细胞或组织的细胞核染色。
使用说明:
1. 对于固定的细胞或组织:
a. 对于细胞或组织样品,固定后,适当洗涤去除固定剂。随后如果需要进行免疫荧光染色,则先进行免疫荧光染色,染色完毕后再按后续步骤进行Hoechst 33342染色。如果不需要进行其它染色,则直接进行后续的Hoechst 33342染色。
b. 对于贴壁细胞或组织切片,加入少量Hoechst 33342染色液,覆盖住样品即可;对于悬浮细胞,至少加入待染色样品3倍体积的染色液,混匀。室温放置3-5分钟。
c. 吸除Hoechst 33342染色液,用TBST、PBS或生理盐水洗涤2-3次,每次3-5分钟。
d. 直接在荧光显微镜下观察或封片后荧光显微镜下观察。细胞发生凋亡时,会看到凋亡细胞的细胞核呈致密浓染,或呈碎块状致密浓染。
2. 对于活细胞或组织:
a. 加入适当量Hoechst 33342染色液,必须充分覆盖住待染色的样品,通常对于六孔板一个孔需加入1ml染色液,对于96孔板一个孔需加入100微升染色液。
b. 在适宜于细胞培养的温度下培养20-30分钟。弃染色液,用PBS或培养液洗涤2-3次即可进行荧光检测。
注意事项:
荧光染料都存在淬灭的问题,为减缓荧光淬灭可以使用抗荧光衰减封片剂。建议染色后尽量当天完成检测,活细胞或组织染色后应立即观察。
为了您的安全和健康,请穿实验服并戴一次性手套操作。
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