Ab69506

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.

Protein from each right hemisphere sample was extracted, quantified, and denatured at 95°C in 5X loading buffer for 10 min. Western blot was performed as previously described (17 (link)). The following primary antibodies were used: Fibrin (ogen) (ab34269, Abcam), P-selectin (60322-1-Ig, Proteintech), PDGFRβ (ab69506, Abcam), CypA (ab41684, Abcam), N-cadherin (22018-1-AP, Proteintech), phospho-NF-κB p65 subunit (p-p65, ab86299, Abcam), MMP-9 (10375-2-AP, Proteintech), and β-actin (66009-1-Ig, Proteintech). The bands were visualized using a BeyoECL Plus kit (P0018; Beyotime) and photographed by a chemiluminescence imaging system (ChemiDoc XRS+; Bio-Rad, Hercules, CA, USA). Band densities were quantified by a blinded observer using ImageJ software.

The SHEDs, DPSCs, and pericytes were cultured in MesenCult™ MSC Basal Medium (Cat.05401; STEMCELL Technologies) supplemented with MesenCult™ MSC Stimulatory Supplement (Cat. 05402S; TEMCELL Technologies) for 2 weeks. Subsequently, the cells were fixed with 4% (w/v) cold paraformaldehyde (PFA) for 15 min and washed with PBS. The cells were then permeabilized using 0.1% (v/v) Triton-X100 (Sigma-Aldrich) for 10 min. Primary antibodies against NG2 (LS-C72310; LSBio, Seattle, WA, USA), DESMIN (ab15200; Abcam), and PDGFR-β (ab69506; Abcam) were used for immunocytochemical staining. Goat anti-rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150077; Abcam) and goat anti-mouse IgG H&L (Alexa Fluor® 488) (ab150117; Abcam) were used as secondary antibodies. The images were captured under fluorescence (Olympus BX60; Olympus, Center Valley, PA, USA) and confocal laser scanning microscopy (LSM 510-META; Carl Zeiss, Jena, Germany).