Ab157107

Coronal cryosections (12 μm) of rat brains were obtained from the center of the anterior-posterior extent of the tumor. Tissues were blocked for 1 h in 2% donkey serum with 0.2% Triton X-100 and labeled overnight at 4°C with primary antibodies against GFAP (1:500; C9205, Sigma-Aldrich), ED-1 (1:200; MAB 1435, Millipore, Billerica, MA), RECA-1 (1:200; MA1-81510, Thermo Fisher Scientific Inc.), HIF-1 (1:200; sc-12542, Santa Cruz Biotechnology, Inc.), CD44 (1:200; ab157107, Abcam), Olig2 (1:100; ab109186, Abcam), CD3 (1:200; ab5690, Abcam), CD8a (1:200; ab33786, Abcam), CD4 (1:200; sc-1573, Santa Cruz Biotechnology, Inc, CD49d (1:200; ab22858, Abcam), P2Y12 (1:200; AS-55043, AnaSpec, Fremont, CA) or IBA1 (1:100; ab5076, Abcam). Alexa Fluor 500- or fluorescein isothiocyanate-conjugated secondary antibodies were applied, and tissues were coverslipped with ProLong Gold antifade reagent (P36930; Thermo Fisher Scientific Inc.). Specific labeling was confirmed by omission of primary antibody. Immunolabeled tissues were visualized with epifluorescence microscopy (Nikon Eclipse 90i; Nikon Instruments Inc., Melville, NY).

The ENO1 antibody (ab227978), β-Tubulin antibody (ab52901), anti-CD44 antibody (ab157107), SOX2 antibody (ab97959), anti-Nanog antibody (ab80892), and anti-Oct4 antibody (ab18976) were from Abcam.

For immunoprecipitation of CD44, 5×10⁶ CL1-5/OPN-a cells were lysed with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 2 mM EDTA 2, 1 mM PMSF, protease inhibitor, and phosphatase inhibitor). The lysate (1 mg protein) was incubated with 4 μg anti-CD44 (Abcam, ab157107) antibody overnight at 4°C. Washed protein G beads (50 μL, MagQu, MF-PRG-3000) were then added to the protein-antibody mixture followed by incubation for 5 h at 4°C. After washing, the protein complex was recovered by heating at 95°C for 3 min in loading buffer. CD44 and OPN were resolved with SDS-PAGE and immunoblots were subsequently probed with anti-CD44 (Abcam ab157107) and anti-OPN (O17, IBL 18625) antibodies.

hDPSCs were fixed with 4% paraformaldehyde for 15 min and blocked with normal goat serum at room temperature for 30 min. The cells were then incubated with primary antibodies against CD44 (ab157107, 1:500; Abcam, Cambridge, UK), CD90 (ab133350, 1:100; Abcam), CD105 (ab221675, 1:1,000; Abcam, Cambridge, UK), CD29 (ab24693, 1:1,000; Abcam, Cambridge, UK), CD45 (ab10558, 1:1,000; Abcam, Cambridge, UK), and CD11b (ab8878, 1:200; Abcam, Cambridge, UK) overnight at 4°C. After cells were washed with PBS solution, they were re-probed with Alexa Fluor 488-labeled goat anti-rabbit immunoglobulin G (IgG) (ab150077, 1:200; Abcam) under complete darkness at 37°C for 1 h. The cells were subsequently left to stain with 5 μg/mL 4′,6-diamidino-2-phenylindole for 5 min and mounted at 4°C in complete darkness. The cells were then observed under a laser-scanning confocal microscope (ZEISS LSM 510 M ETA; Carl Zeiss, Jena, Germany) and imaged using NIS-Elements Viewer software at an emission wavelength of 519 nm. The nuclei were stained in blue fluorescence, whereas cells positive for CD44 and CD90 were stained in green fluorescence.

Skin sections were stained with an anti-GFP antibody. The skin sections were also treated with primary Abs against CD31 (Abcam, ab28364, diluted 1:300), vWF (Abcam, ab11713, diluted 1:300), CK14 (Abcam, ab7800, diluted 1:500), CD44 (Abcam, ab157107, diluted 1:400), E-selectin (Abcam, ab18981, diluted 1:400), Secondary antibodies conjugated to rhodamine-isothiocyanate were used for fluorescence detection on a confocal laser scanning fluorescence microscope (Zeiss, German).

Frozen tumors from the animal studies were cryopreserved in 30% sucrose solution and then embedded in OCT (Tissue-Tek, Torrance, CA, USA). 12 μm thick tumor sections (Leica CM1950 Cryostat, Leica Biosystems, Deer Park, IL, USA) were fixed in 4% paraformaldehyde (PFA) and incubated in goat serum buffer for 1 h. After washing with PBS, primary antibodies were: CD44 (Rabbit, 1:50, Abcam, ab157107), and CD166 (Rabbit, 1:50, Abcam, ab109215), and secondary antibody was goat anti-rabbit-Alexa Fluor488 (1:1000, Cell signaling, Danvers, MA, USA). Sections were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were acquired using a Fluor Motorized DIC Polarization Phase Contrast Microscope (Zeiss AXIO Observer) with consistent intensity and exposure parameters at 400× magnification.