Ab56299

To detect blood vessels and macrophages in the connective tissue of tooth extraction sockets, rat anti-mouse CD31 monoclonal antibody (ab56299; Abcam, Cambridge, MA; 1:100 dilution), rat anti-mouse F4/80 monoclonal antibody (ab16911; Abcam; 1:50 dilution), and rabbit anti-mouse CD206 polyclonal antibody (ab64693; Abcam; 1:50 dilution) were used as primary antibodies. Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 goat anti-rat IgG (Invitrogen, Carlsbad, CA; 1:200 dilution) were used as secondary antibodies. Sections were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA). Stained sections were photomicrographed by fluorescence microscopy (Axio Scope A1; Zeiss) and were histomorphometrically analyzed with ZEN2 software and NIH ImageJ to yield the following parameters: the number of blood vessels [blood vessels (#/mm²)]; vessel surface area in the AOIs of soft tissue in the tooth extraction sockets [vessel density (%)] (AOI, 200 × 1000 μm); F4/80⁺CD206⁻ macrophages [F4/80⁺CD206⁻ cells]; and F4/80⁺CD206⁺ macrophages [F4/80⁺CD206⁺ cells] in the AOIs of soft tissue in the extraction sockets (AOI, 200 × 1000 μm).

The presence of osteoblasts was detected by immunohistochemistry using an antibody specific for human osteocalcin (M184, Takara Bio Inc., Japan). To determine the origin of blood vessels in the re-vascularised bone implants, a dual immunofluorescence protocol using species-specific CD31 antibodies was performed to detect endothelial cells; human-specific CD31 (1:400, ab76533, Abcam, UK) and mouse-specific CD31 (1:400, ab56299, Abcam, UK). To establish whether genetic alterations observed between PDXs growing in the fat pad and those that had metastasised to bone were translated into changes in protein expression immunohistochemistry for IL-1B (1:200, ab2105, Abcam), IL1R1 (1:200, ab154524, Abcam), S100A4 (1:500, ab40722, Abcam, hRAS (1:200, ab97488, Abcam), DKK (1:200, Ab38594, Abcam), Gamma Catenin (1:25, 2309, Cell signalling), Fibronectin (1:50, ab32419, Abcam). Staining was visualised with corresponding biotin-conjugated secondary antibodies (Vector Laboratories, 1:200) and either avidin FITC (mouse) or avidin TRITC (human) (Vector Laboratories).

After one hour of acoustic activation and microbubble navigation in the mouse, mice were euthanized through an intraperitoneal (i.p.) injection of Pentobarbital (Sigma-Aldrich, Cerilliant®) at an overdose level (200 mg/kg), followed by decapitation. The brains were then extracted and sliced into coronal sections, each 1 mm thick, spanning from 6.5 to 0.5 mm anterior to the inter-aural line. The antibodies that were used for histology were: anti-CD31 (cat. # ab56299, Abcam, 1:100), anti-NeuN (cat. # AB177487, Abcam, 1:1500), and anti-GFAP (cat. # ab4674, Abcam, 1:2000). Tissue sections were incubated with DAPI (4’,6-diamidino-2-phenylindole dihydrochloride) solution (stock 10 mg/ml, 1:10,000, cat. #D9542, Sigma-Aldrich,). See Supplementary Fig. 25.

Paraffin sections (4 µm) of TC-1 tumors were dewaxed in xylene and rehydrated through graded ethanol to water. Antigens were retrieved by boiling in 10 mM citrate buffer (pH 6.0) for 30 min. The cooled sections were incubated in DAKO REAL Peroxidase-Blocking solution (DAKO, Carpinteria, CA, USA) for 10 min to quench endogenous peroxidase. To block nonspecific binding, sections were incubated in DAKO Protein Blocking solution (DAKO) for 10 min at room temperature. Sections were then incubated with a rabbit polyclonal antibody against mouse MMP-9 (PAB12714, Abnova, 1∶100 dilution) in DAKO REAL Antibody Diluent (DAKO) overnight at 4°C. The slides were incubated for 1 hour at room temperature with peroxidase-conjugated secondary antibodies, washed, incubated with DAB, counterstained with hematoxylin, dehydrated through an ethanol series and xylene, and mounted. To evaluate tumor microvessel formation, tumor sections were stained for CD-31 using a rat monoclonal antibody against mouse CD-31 (ab56299, Abcam, Tokyo, Japan, 1∶100 dilution).

Slides (5 μm thick) were autoclaved in Target Retrieval Solution (Dako, S1699, Glostrup, Denmark), incubated in Proteinase K (S3004, Dako) or with EDTA-buffer (Prosan) according to the immunolabelling for Ki67, CD31, and Pimonidazole, respectively. Endogenous peroxidases were blocked by 3% H₂O₂/H₂O (Merck) for 20 minutes, and nonspecific binding was prevented by incubation in PBS/Bovine Serum Albumin 10% (Fraction V, Acros Organics, NJ). Tumor sections were incubated with a mouse monoclonal anti-human Ki-67 antibody (1/100) (clone MIB-1, M7240; DAKO), a rat anti-CD31 antibody (1/100) (Ab56299, Abcam), or a mouse monoclonal anti-pimonidazole antibody (1/50) (Hydroprobe-1 MAb-1 clone 4.3.11.3). After 3 washes in PBS or Tris-HCl for CD-31 staining, slides were incubated with a HRP-conjugated secondary antibody, after post antibody blocking (DPVB Blocking, Immunologic NL) for pimonidazole staining, and revealed with Vector DAB (SK-4100, Vector Laboratories, Burlingame, CA, USA). Slides were counterstained with haematoxylin.

All staining was performed according to the manufacturer’s instructions, using 10⁶ cells and the recommended amount of antibodies, followed by incubation for 30 min at room temperature. Briefly, the cells were incubated separately with the primary antibody rat anti-mouse CD31 (ab56299, 1:50; Abcam, Burlingame, CA, USA) for 30 min. After washing 3 times with PBS, the cells were incubated with the secondary antibody goat anti-rat Alexa Flour 488 (A-11006, 1:1,000; Invitrogen). For the negative controls, the cells were incubated only with the secondary antibody. The expression level of CD31 was determined using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).