[Effect of fixation methods on immunocytochemical localization of NMDAR1 on cultured cortical neuron membrane].

ABSTRACT

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 degrees Celsius pure acetone and -20 degrees Celsius 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 (11) 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 degrees Celsius pure acetone and -20 degrees Celsius pure methanol for 5 min. NMDAR1 distributes on the polar membrane of the neurons where the dendrites originate and on the dendritic stem.