Ab182744

All tumor tissue staining experiments were performed at the MD Anderson Cancer Center Research Histology Core Laboratory (28 (link)). Tumors were fixed in 10% neutral buffered formalin, paraffin embedded, processed into a 5 μm thick section/slide, and stained with H&E or various antibodies for IHC. For IHC, the slides were deparaffinized, treated with antigen retrieval buffer, blocked with Sniper (catalog BS966, Biocare Medical), stained with primary and the corresponding secondary antibodies, visualized by diaminobenzidine, and counterstained with hematoxylin. The primary antibodies used were as follows: human-specific cytokeratin (CK) 8/18 (catalog M3652, Dako), collagen VI (catalog ab182744, Abcam), thyroglobulin (TG) (catalog BSB 2767, BioSB), CEACAM5/6 (catalog ab22705, Abcam), paired box gene 8 (PAX8) (catalog 379, Biocare), and thyroid transcription factor 1 (TTF-1) (catalog IS05630-2, Dako). Slides were then examined microscopically by a head and neck pathologist using a BX41 Olympus microscope and an Aperio (Leica Biosystems) digital image scanner. The morphology, degree of differentiation (growth pattern, cytologic features, formation of keratin), and extent of inflammation were evaluated from H&E staining results. The IHC staining intensity was graded according to 3 levels: weak, intermediate, and strong.

Isolation of proteins from cells and tissues was performed as described [37 (link)]. NS0-expressed murine DCN was from R&D Systems, Minneapolis, MN, USA, #1060-DE. Recombinant Human MMP-14/MT1-MMP Protein (918-MP-010) was from R&D Systems. His₆-tagged ΔDCN cloned into the pET28a vector with ₆His at the N-terminus was purified with the His-Tag kit (Novagen, Burlington, MA, USA) as described [30 (link)]. For Western blotting, lysates were resolved on SDS-PAGE, blotted onto an Immobilon-FL membrane (Millipore, Burlington, MA, USA), blocked with Odyssey blocking buffer (LI-COR), and probed (in PBS/0.05% Triton X-100) with antibodies as follows: goat anti-mouse DCN from R&D Systems (#BAF1060) at 1:1000; rabbit monoclonal anti-Col6A1 (Abcam, Cambridge, MA, USA; ab182744) at 1:1000; rabbit polyclonal anti-Col6A1 (Abcam ab6588) at 1:1000; rabbit polyclonal anti-Co1A1 (Abcam #34710) at 1:1000; Rabbit anti-endotrophin antibody [38 (link)] at 1:1000; Rabbit anti-GFP (Abcam ab290) at 1:1000; and anti-β-Actin (Abcam, Cat. # ab8226, 1:5000). The signal was detected using the Odyssey CLx imaging system (LI-COR, Lincoln, NE, USA).

Biphasic scaffolds, both before and after decellularization (n = 3), were rinsed in PBS then fixed for 30 min in 4% paraformaldehyde (PFA) in PBS. The scaffolds were then incubated for 60 min in blocking buffer (1% bovine serum albumin; 10% goat serum; 0.05% tween-20 solution in PBS) at room temperature (RT) and subsequently incubated for 1 h at RT with collagen I mouse primary antibody (1:200, ab6308, Abcam, Melbourne, VIC, Australia) diluted in blocking buffer or collagen VI rabbit primary antibody (1:200, ab182744, Abcam) diluted in blocking buffer. Samples were then washed 3 times with PBS and incubated for 60 min with their respective secondary antibodies; goat anti-mouse IgG Alexa Fluor^® 488-conjugated secondary antibody (1:500, ab150113, Abcam) or donkey anti-rabbit IgG Alexa Fluor^® 647-conjugated secondary antibody (1:500, ab150075, Abcam) diluted in blocking buffer. All samples were then counterstained with 5 μg/mL 4,6-diamino-2-phenylindole (DAPI, Life Technologies, NY, USA) for 10 min at RT to visualise remnant DNA. The scaffolds were then rinsed in PBS and imaged using a confocal microscope (Nikon, Eclipse-Ti, Melville, NY, USA) at an excitation/emission wavelength of 405/417-477 for DAPI, 488/500-550 nm for collagen I (green) and 561/570-1000 nm for collagen VI (red).