Anti map2 sc 20172

The commercially available antibodies used were as follows: rabbit anti-BACE1 (5606), anti-nicastrin (9447S), anti-GGA3 (8027), anti-rab5 (3547), anti-rab7 (9367), and anti-rab9 (5118) from Cell Signaling Technology; anti-APP C-term (recognizes C-terminal part of APP, 18961), anti-APP N-term (10D1), and anti-sAPPβ-sw (10321, clone 6A1) from Immuno-Biological Laboratories; anti-Aβ (SIG-39220, clone 4G8) and anti-sAPPα (SIG-39320, clone 6E10) from SIGNET; anti-Lamp1 (ab25630) and anti-PSD95 (ab2723) from Abcam; anti-actin (A4700) from Sigma; anti-syntaxin 6 (610635) from BD Biosciences; anti-GAPDH (MAB374), anti-APP (22C11), and anti-MBP (MAB386) from Millipore; anti-MAP2 (sc-20172) from Santa Cruz Biotechnology; anti-GFAP (13-0300) from Life Technologies; anti-CHL1 (AF2147) and anti-contactin-2 (AF4439) from R&D systems; and anti-Iba1 (019-197419) from Wako. Biotinylated erythroagglutinating phytohemagglutinin (E4-PHA) lectin was from Seikagaku Corporation.

Cells were fixed with 4% paraformaldehyde (PFA; P6148, Sigma-Aldrich) for 20 min at room temperature (RT). Blocking was performed with blocking solution: 10% Fetal Bovine Serum (FBS) or 5% Goat serum (GS) with 0.2% Triton X 100 in PBS for permeabilization. Cells were then incubated with primary antibodies (anti-SSEA4, CST-4755, Cell Signaling Technology, Danvers, MA, USA; anti-TRA-1-60, ab16288, Abcam, Cambridge, UK; anti-OCT4, sc-5279, Santa-Cruz, Starr County, TX, USA; anti-FMRP, BLG-834601, Biolegend, San Diego, CA, USA; anti-Tuj1, BLG-801201, Biolegend; anti-PAX6, BLG-901301, Biolegend; anti-MAP2, sc-20172, Santa Cruz; anti-SYN1, AB1543, Merck, Darmstadt, Germany; anti-PSD-95, MAB1596, Merck) diluted in blocking solution for 1 h at RT, washed 3 times with PBS, and incubated with secondary antibodies (donkey anti-mouse Alexa Fluor 488, A21202, Thermo Fisher Scientific, Waltham, MA, USA; goat anti-rabbit Alexa Fluor 594, A11012, Thermo Fisher) for another 1 h at RT in the dark, and counterstained with DAPI for nucleus localization (D1306, Thermo Fisher Scientific). Cells were mounted with Fluoromount aqueous mounting medium (00-4958-02, Thermo Fisher Scientific). Bright-field, phase, and fluorescence images of cells were obtained using an Olympus IX51 inverted light microscope (Olympus, Tokyo, Japan).