Neuronal nuclei (neun)

A second set of hippocampal brain sections cut at 20 µm were processed for Iba‐1 (Cat. # 019‐19741 WAKO Chem.) and NeuN (Abcam prod. # 134014) immunofluorescent staining. Sections were processed with a mounted protocol. After mounting, hippocampal sections were immersed in 0.05 M glycine in 0.3% Triton in PBS (T‐PBS) for 30 min to reduce autofluorescence, followed by rinses in 0.3% T‐PBS, followed by incubation in 15% bovine serum albumin (BSA) in 0.3% T‐PBS blocker, and finally primary antibody cocktail containing 15% BSA and 0.03% T‐PBS overnight at 4°C on a rocker. Primary antibodies were diluted at 1:500 concentration in solution. The following day, sections were washed in 0.03% T‐PBS, incubated in fluorescent secondary antibody cocktail 15% BSA and 0.03% T‐PBS for 2 hr. Secondary antibodies included Alexa Fluor goat anti‐chicken 568 (cat# A‐11041, Life Technologies, Thermo Fisher Sci.) and Alexa Fluor goat anti‐rabbit 488 (cat# A‐11008, Life Technologies, Thermo Fisher Sci.) at a 1:250 concentration in solution. Sections were washed with 0.03% T‐PBS, then PBS, and finally were cover‐slipped with VectaShield^® mounting media. Slides were kept at 4°C in the dark until imaged.

Prior to proceeding with the IHC/IF protocol, a standard process of deparaffinizing sections was performed in xylol and alcohol series. Antigen retrieval was performed by boiling sections in citrate buffer (pH 6,0). After three washes in PBS, blocking solution containing 1–3% BSA and 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MI, USA) in PBS was applied on the sections for 1–2 h. The blocking solution was replaced with primary antibodies diluted in a blocking solution and incubated overnight at 4 °C. The following antibodies were used: NeuN (Abcam, Cambridge, UK), MAP2 (Sigma-Aldrich, St. Louis, MI, USA), MAP2 (Merck Millipore, Burlington, MA, USA), CUX2 (Abnova, Taipei, Taiwan), CUX2 (Abcam, Cambridge, UK), Reelin (Millipore), DCX (Merck Millipore, Burlington, MA, USA), FOXP1 (Abcam, Cambridge, UK), Neuroserpin (Abcam, Cambridge, UK), TLE4 (Santa Cruz Biotechnology, Dallas, TX, USA), Nurr1 (R&D systems, Minneapolis, MN, USA). After incubation, sections were washed in PBS, and appropriate secondary antibodies (Alexa Fluor, Thermo Fisher Scientific, Waltham, MA, USA) were applied for 2 h at RT. Following three washes in PBS, TrueBlack quencher (Biotium, Fremont, CA, USA) was applied on the sections. Finally, the sections were covered using a mounting medium with DAPI (Vectorlabs, Burlingame, CA, USA). The IHC protocol was implemented as previously described [32 (link)].