Ab28364

Cells were lysed using a RIPA buffer and processed as previously described [22 (link)]. The following antibodies were used: anti-Vimentin (Abcam #ab8978), anti-N-Cadherin (Abcam #ab18203), anti-GAPDH (Abcam # ab8245), anti-β-Actin (Abcam #ab8227), anti-CD31 (Abcam #ab28364), and anti-IL-3Rα/CD123 (R&D Systems #MAB301-100). Secondary antibodies conjugated with peroxidase were purchased from Cell Signalling Technologies (Danvers, MA, USA). Supplementary Information reports the details.

Histology was performed as described before^{35 (link)}. Primary antibodies used were Plin2: Fitzgerald # 20R-AP002 diluted 1:500; CD31: Abcam #ab28364 diluted 1:160; Gpnmb: R&D #AF2550 diluted 1:100; Lgals3: Enzo lifescience #CLO49P, diluted 1:200 in PBS. Details about the procedure can be found in the SI Methods.

The brains of the mice were removed and fixed in PFA at 4°C overnight. Subsequently, they were transferred to a sucrose solution of gradually increasing concentration for 48–72 h. The brain samples were then sliced into 20 μm thick coronal sections using a CryoStar™ NX50 Cryostat (Thermo Fisher Scientific, Waltham, USA). For immunochemistry, the brain sections were incubated overnight with primary antibodies including anti-NeuN, anti-GFAP, anti-IBA1, anti-CD31 and anti-TuJ1 (Abcam, ab104224, ab7260, ab15690 and ab28364, and R&D, MAB1195) at 4°C overnight. The sections were rinsed with phosphate buffered saline (PBS) twice, followed by incubation with the corresponding secondary antibodies (Thermo Fisher Scientific, A11008, A11001) at room temperature for 2 h and then rinsed with PBS. Finally, the slices were covered with DAPI (ZLI-9557, ZSGB-BIO, China) and images were captured using a Zeiss fluorescence microscope (LSM 800, Germany).

Sections were washed three times with PBS followed by antigen retrieval in 0.25% Triton for 10 min. Samples were blocked for 1 h in 10 % goat serum followed by incubation with primary antibodies for 2 h at room temperature (RT). Antibodies against α-sarcomeric Actinin (1/400, Sigma, A7811), α-smooth muscle actin (1/100, Sigma, A2547), CD31 (1/100, Abcam, ab28364), DDR2 (1/100, R&D, MAB25381), WT1 (1/100, Abcam, ab89901), and PDGFRα (1/100, Santa Cruz, sc338) were used. Alexa fluor 647 secondary antibodies were used (1:100, Invitrogen) for 1 h at RT. Co-localization of proteins with Rainbow-labeled clones was obtained through confocal z-stack analysis. WGA (1/500, Invitrogen) staining was performed for 1 h at RT. For co-localization purposes, blue pseudocolor was assigned to all clones analyzed in this manuscript.

The cells were plated on coverslips and allowed to grow before being fixed with 4% PFA at room temperature for 30 min. Subsequently, the coverslips were blocked with PBS containing 5% donkey serum at room temperature for 30 min. The cells were then incubated with primary antibodies overnight at 4 °C. Following the removal of the primary antibodies, the sections were washed in PBS and stained with secondary antibodies for 45 min at room temperature. The nuclei were counterstained with DAPI before mounting the coverslips on glass slides.
The following primary antibodies were used for immunostaining: CD31 (Abcam, ab28364, 1:100 for coverslips or R&D Systems, FAB3628G, 1:100 for bone sections), Endomucin (Santa Cruz, sc-65495, 1:200), alpha smooth muscle actin (Abcam, ab124964, 1:400), CD45 (BD Pharmingen, 557659, 1:200), and RUNX2 (Cell Signaling Technology, 12556 S, 1:400).
The following secondary antibodies were used in immunostaining (all obtained from Jackson ImmunoResearch): donkey anti-rabbit Alexa Fluor 488 (711-545-152, 1:400) and Alexa Fluor 647 (711-605-152, 1:400); donkey anti-rat Alexa Fluor 488 (712-545-150, 1:400), Alexa Fluor 594 (712-585-150, 1:400), and Alexa Fluor 647 (712-605-150, 1:400); and donkey anti-goat Alexa Fluor 488 (705-545-147, 1:400).