Igg1 mouse pe

NPC surface markers were analyzed by flow cytometry as described previously [48 (link)]. After PQ treatment, cells were washed three times with PBS, and incubated on ice for 1 h in the dark with antibodies against APC-conjugated Tie-2 (FAB3131A, R&D Systems, MN, USA), PE-conjugated GD2 (562,100, BD Biosciences), and FITC-conjugated CD24 (555,427, BD Biosciences). For the isotype control, the antibody was replaced with IgG1 mouse APC (IM2475, Beckman Coulter, CA, USA), IgG1 mouse PE (A07796, Beckman Coulter), and IgG1 mouse FITC (A07795, Beckman Coulter). Only viable cells were analyzed using the PI-negative gate.
For ECM analysis, cells were fixed and permeabilized with IntraPrep Permeabilization Reagent (A07803, Beckman Coulter). Cells were then subjected to three cycles of freezing (−80 °C) and thawing (37 °C). Subsequently, cells were incubated overnight at 4 °C with primary antibodies against collagen II (NB600-844, Novus Biologicals, CO, USA) and proteoglycan (MAB2015, Sigma-Aldrich). After washing with PBS, cells were reacted with goat anti-mouse Alexa Flour 488-conjugated secondary antibody (A28175, Invitrogen) on ice for 1 h in the dark, and then analyzed by flow cytometry.