β tubulin 3 antibody

The cells were assigned to the following groups: control group, ketamine group, and 17β-estradiol group. No drug treatment was added to the control group. NSCs in the ketamine group were exposed to 100 μM ketamine for 24 h. NSCs in the 17β-estradiol group were pretreated with 17β-estradiol (100 nM) for 30 min and then 100 μM of ketamine was added to the culture medium for 24 h. For proliferative analysis, NSCs were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with BrdU for the last 4 h. Following being fixed with 4% paraformaldehyde, the cells were stained with BrdU antibody (1:200, Abcam, United Kingdom) and DAPI. As for neuronal differentiation analysis, after being exposed to ketamine with or without 17β-estradiol for 24 h, the cells were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with differentiating medium for 7 days, then the cells were harvested for immunohistochemical staining. The cells were labeled with β-tubulin III antibody (1:500; Sigma-Aldrich Inc. St. Louis, MO, United States). Briefly, 5–7 randomly selected fields were captured in each coverslip, and the numbers of β-tubulin III-positive cells were counted (at least 200 cells per test case). Data were collected from three independent experiments.

Roscovitine-treated and untreated neurons were washed with 1× Phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Invitrogen Life Technologies), stained with β-tubulin III antibody (1:200 dilution; Sigma-Aldrich Co.) in 1× PBS containing 10% goat serum and 0.3% Triton X-100 and Cy3-conjugated anti-mouse secondary antibody (1:200 dilution; Sigma-Aldrich Co.) in 1× PBS containing 10% goat serum and 0.3% Triton X-100. Images were captured under a Zeiss fluorescence microscope. Neurite length was analyzed by the MetaXpress software and by an observer blinded to their condition (Molecular Devices, Sunnyvale, CA, USA). Between 40 and 60 neurons were analyzed per condition. Three to four rats per condition were routinely used. Graph Pad Prism was used for statistical analysis. Student’s t-tests were carried out with the statistical significance set at p ≤ 0.05.

Cell culture reagents were from PAA. Purified LTA was from Invivogen; LPS, apyrase and reactive blue 2 (RB2), were from Sigma, authentic peroxynitrite was from Cayman, 3-morpholinosydnonimine (SIN-1) was from Invitrogen, MRS2578, MRS2693, and UDP were from Tocris, Amyloid β_1-42 peptide was from EZBiolab, Annexin V was from BioVision. Tumor necrosis factor-α and interleukin-1β Quantikine Elisa Kits were from R&D Systems. NeuN antibody was from Millipore, β-tubulin III antibody was from Sigma, anti-cleaved caspase-3 antibody was from Cell Signaling Technology, Alexa Fluor 488-labeled Griffonia simplicifolia isolectin B₄ and secondary goat-anti-mouse-Alexa Fluor 488 antibody were from Invitrogen, secondary goat anti-mouse-Cy3 and goat anti-rabbit-Cy3 antibodies were from Jackson ImmunoResearch Laboratories. All other materials were purchased from Sigma.