Fluorescent staining
This protocol is extracted from research article:
Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture
Cannabis Cannabinoid Res, Feb 12, 2021; DOI: 10.1089/can.2019.0102

Neurons were treated with the Neurobasal media containing CBD (1, 5, or 10 μM) or 0.1% DMSO at 1 DIV. After incubation for 24 h, the neurons were treated with Celltracker Red CMTPX Dye at 37°C for 30 min, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, and washed three times with PBS each for 5 min. The fixed samples were permeabilized with 0.1% Triton X-100 solution in PBS for 10 min at room temperature and washed three times with PBS each for 5 min. The samples were then treated with Alexa Fluor 488 phalloidin for visualizing F-actin or rabbit-produced β-tubulin Ⅲ primary antibody, incubated at 37°C, and washed with PBS three times each for 5 min. For visualizing β-tubulin Ⅲ, the samples were additionally treated with Alexa Fluor 594 goat anti-rabbit secondary antibody. Finally, the samples were mounted on slide glasses with the DAPI solution to stain nuclei for 30 min. Fluorescence images were obtained with an LSM 700 confocal microscope.

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