Primary Cortical Neuron Immunocytochemistry

Primary cortical neuronal cultures derived from C57Bl/6 and Tau KO mice (Tau KO primary neurons only used for LDH) were prepared and maintained as previously described [74 (link), 84 (link), 92 (link)]. The primary antibodies used in this study for immunocytochemistry are as follows: rabbit anti-I11 [55 (link)], mouse anti-β-III-Tubulin (Abcam #78078), rabbit anti-total p53 (Abcam #246550), rabbit anti-total tau (Abcam #64193), mouse anti-phosphorylated p53 (Ser15) (Cell Signaling #9284), and rabbit anti-phosphorylated histone H2AX (Ser139) (Cell Signaling #2577). After three washes with PBS, cells were probed with mouse and rabbit-specific fluorescent-labeled secondary antibodies (1:200, Alexa Fluor 568, Life Technologies). The single-frame images and Z-stacks for orthogonal view were collected using a Keyence Confocal Microscope. To build the Z-stack, 17 stacks/0.3–0.4-μm optimal thickness were captured. Each treatment condition was randomly imaged in five different regions of interest and performed in duplicate. Imaging processed with ImageJ Software (NIH).