Ab7260

For the 2D culture, cells were fixed in 4% paraformaldehyde (PFA) for 15 min, blocked in 5% BSA with 0.05% Triton-X, and incubated overnight with primary antibody against type I collagen, α-SMA, GFAP and ki67 (1:1000; AbCam, Cambridge, MA, USA; catalog #ab34710, #ab5694, #ab7260, and #ab15580, respectively), Gata4 and Reelin (1:100; Santa Cruz, Dallas, TX, United States; catalog #sc-1237 and #sc-25346, respectively), followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Invitrogen™, Waltham, MA, USA; catalog #A-21206 and #A-11056). For the 3D culture, organoids were fixed in 4% PFA for 2 h, dehydrated in 30% sucrose overnight, embedded into Tissue Tek^® O.C.T. Compound, and sliced at 10 μm thickness in cryostat microtome. Slides with sectioned organoids were processed for antibody staining using the same method as cells from the 2D culture and preserved in Fluoromount-G™ Mounting Medium with DAPI (Invitrogen™; catalog #00-4959-52). Additional primary antibodies used for organoid studies include anti-albumin antibody (1:200; Bethyl, #A80-129A) and anti-PDGFRβ (1:1000, AbCam, catalog #ab69506). All IF samples were visualized by EVOS M7000 cell imaging system (Invitrogen™), and images at each fluorescent channel were taken at equivalent exposure for comparison.

Hippocampal tissue samples were obtained on post-surgical day 30. Membrane protein isolation was performed using the Mem-PER Plus^TM membrane protein extraction kit (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions. The following primary antibodies were used: polyclonal rabbit anti-GABA_A-α1 subunit (1:500; Abcam, catalog #ab7260), monoclonal mouse anti-hnRNPA2/B1 (1:200; Santa Cruz Biotechnology, catalog #sc-10035), monoclonal mouse anti-amyloid β (1:2,000, Sigma-Aldrich, United States), and monoclonal mouse anti-β-actin (1:10,000, Proteintech, United States). Image-Pro Plus was used for image acquisition and analysis.

The primary antibodies used for Immunofluorescence staining in this study were anti-CD31 (1:300, Abcam, ab222783), anti-ZO-1 (1:300, Abcam, ab221547), anti-Occludin (1:300, Abcam, ab216327), anti-NeuN (1:300, Abcam, ab104224), anti-GFAP (1:1000, Abcam, ab7260), anti-Sox17 (1:100, Santa Cruz Biotechnology, sc-130295), and anti-CD68 (1:300, Abcam, ab283654). The secondary antibodies used for immunofluorescence staining in this study were Alexa Fluor® 488 AffiniPure™Goat Anti-Rabbit IgG (H + L)(1:300, Jackson, 111-545-003) and Alexa Fluor® 594 AffiniPure™Goat Anti-Mouse IgG (H + L)(1:300, Jackson, 115-585-003). Primary antibodies used for western blot studies were anti-ZO-1 (1:1000, Abcam, ab221547), anti-Occludin (1:1000, Abcam, ab216327), anti-β-actin (1:2000, Servicebio, Wuhan, China), anti-Sox17 (1:500, Santa Cruz Biotechnology, sc-130295; 1:1000, Proteintech, Wuhan, China, 24903-1-AP), anti-CD31(1:1000, Abcam, ab222783), anti-UCHL1 (1:1000, Abcam, ab108986), anti-Flag-Tag (1:2000, Cell Signaling Technology, #14793), anti-Myc-Tag (1:2000, Cell Signaling Technology, #9402), and anti-HA-Tag (1:2000, Cell Signaling Technology, #3724). The secondary antibodies used for western blot studies were HRP conjugated Goat Anti-Rabbit IgG (H + L) (1:5000, Servicebio, GB23303) and HRP conjugated Goat Anti-Mouse IgG (H + L) (1:5000, Servicebio, GB23301).