固定方式对神经细胞膜NMDAR1免疫细胞化学定位的影响

Effect of fixation methods on immunocytochemical localization of NMDAR1on cultured cortical neuron membrane

目的探讨不同固定方式对神经细胞膜NMDA受体NR1亚基免疫细胞化学定位结果的影响,并确定NR1在膜上的亚细胞定位分布。方法采用免疫细胞化学ABC法观察不同固定方式分组时NR1亚基在神经元膜上的定位分布。结果-20 ℃纯甲醇和-20 ℃纯丙酮固定5 min组呈现NR1典型的细胞膜阳性染色,定位在神经元两极靠近树突起始部及树突干上。4%多聚甲醛及加上0.5%戊二醛固定20 min组呈现染色假阴性。95%乙醇固定10 min组呈现细胞膜染色假阴性,细胞核染色假阳性。结论每种抗原免疫细胞化学检测中都有各自最适合的固定方法。NMDAR1抗原需要应用缓和的固定方式,-20 ℃纯甲醇和-20 ℃纯丙酮5 min短时间固定效果最好。NMDAR1分布于神经元膜两端树突起始部及树突干上。

Objective To investigate the changes in subcellular localization of NMDAR1 on cultured cortical neurons in response to different methods for fixation of neuron cells. Methods Subcellular localization of NMDAR1 in cultured cortical neurons fixed with different fixatives and procedures was studied by immunocytochemical avidin-biotin peroxidase (ABC) method. Results Neurons fixed with -20 ℃pure acetone and -20 ℃pure methanol for 5 min presented typical positive staining of NR1 subunit located on the polar membrane of the neurons where the dendrites originated and on the stem of the dendrites. The neurons fixed with 4% formaldehyde alone for 20 min or in the presence of 0.5% glutaraldehyde (1∶1) for 10 min resulted in false-negative staining. Fixation of the neurons with 95% ethanol for 10 min yielded false-negative staining on the membrane and false-positive staining in the nuclei. Conclusions Each antigen may have its specific demand for fixation method in immunocytochemistry, and for NMDAR1 antigen, mild fixation is recommended as by -20 ℃pure acetone and -20 ℃pure methanol for 5 min. NMDAR1 distributes on the polar membrane of the neurons where the dendrites originate and on the dendritic stem.