免疫荧光技术中不同固定剂对细胞p65核移位观察效果的影响

The effects of different fixing agents on the translocation of p65 protein by using immunofluorescence technique

目的观察免疫荧光技术中不同固定剂以及Triton X-100不同渗透时间对小鼠腹腔巨噬细胞p65蛋白核移位的影响,找出最适合观察p65蛋白移位的固定方法及Triton X-100渗透时间。方法采用免疫荧光的方法观察使用不同固定剂(甲醇,1%～4%甲醛)固定,0.1%Triton X-100渗透3～15min的条件下,观察脂多糖刺激2h后,p65蛋白细胞内定位的情况。结果 p65蛋白在对照细胞主要定位于细胞质,脂多糖(LPS)刺激后p65蛋白移入胞核。甲醇固定后细胞荧光染色显色很弱且核区不明显,染色弥散;1%、2%甲醛固定不良可引起细胞收缩;4%甲醛固定后,核区与胞质p65蛋白荧光染色对比明显。0.1%Triton X-100作用3～5min效果最佳,而作用10～15min对照细胞核内也有较强荧光。结论本实验表明,在免疫荧光技术中采用4%甲醛固定20min,0.1%Triton X-100渗透3～5min可更好的观察LPS诱导的小鼠腹腔巨噬细胞p65核移位。

Objective To investigate the effects of different fixing agents and penetration duration time on the observation of p65 protein translocation, and to optimize the fixing and penetrating methods. Methods The translocation of p65 after stimulated by LPS was observed by using immunofluorescence technique. The effects of different fixing agents and various penetration duration of time on observation were compared. Results Untreated cells showed p65 in cytoplasm. LPS induced p65 translocation from cytoplasm to nuclear. P65 staining was much weaker when fixed by methanol. Four percent formaldehyde and 0.01 Triton X-100 penetrating 3-5 min gave the most distinguished staining between cytoplasm and nucleus. Conclusion Four percent formaldehyde and 0. 01 Triton 3：-100 penetrating 3-5 min are suitable for observing LPS induced p65 translocation by using immunofluorescence technique.