Antigen decloaker

Proliferating intestinal epithelial cells were stained with 5-Bromo-2’deoxyuridine (BrdU). At 90 minutes prior to sacrifice, mice received intraperitoneal injections of BrdU (5mg/ml in 0.9% saline, Sigma-Aldrich) to label S-phase cells [23 (link),24 (link)]. Jejunal tissue was then fixed in 10% formalin for 24 hours before being embedded in paraffin and slide-mounted in 5μm sections. Slides were then deparaffinized, rehydrated, and incubated in 1% hydrogen peroxide for 15 minutes before being heated in a pressure cooker in antigen decloaker (Biocare Medical, Concord, CA) for 45 minutes. Protein block (Dako, Carpinteria, CA) was performed for 30 minutes at room temperature and slides were incubated overnight at 4°C with rat monoclonal Anti-BrdU (1:500; Accurate Chemical and Scientific, Westbury, NJ). Samples were then incubated with goat anti-rat antibody (1:500; Accurate Chemical & Scientific) and streptavidin horseradish peroxidase (1:500; Dako), each for an hour at room temperature. Diaminobenzidine (DAB) was used to develop slides for 2–3 minutes, and counterstaining was performed with hematoxylin. BrdU-positive cells were quantified in 100 contiguous, well-oriented intestinal crypts.

Apoptotic cells in the intestinal epithelium were quantified in 100 contiguous and well-oriented crypt-villus units via active caspase-3 staining^{21 ,22 (link)}. Intestinal sections were deparaffinized, rehydrated, and incubated in 3% hydrogen peroxide for 10 minutes. To facilitate antigen retrieval, slides were placed in antigen decloaker (Biocare Medical, Concord, CA) and heated in a pressure cooker for 45 minutes. Sections were blocked with 20% goat serum (Vector Laboratories, Burlingame, CA) and incubated with rabbit polyclonal anti–active caspase-3 (1:100; Cell Signaling Technology, Beverly, MA) overnight at 4 °C. Sections were then incubated with goat anti-rabbit biotinylated secondary antibody (1:200; Vector Laboratories) for 30 minutes at room temperature, followed by Vectastain Elite ABC reagent (Vector Laboratories) for 30 minutes at room temperature. Slides were developed with diaminobenzidine and counterstained with hematoxylin.

Mice were intraperitoneally injected with 5-Bromo-2′deoxyuridine (BrdU, 5 mg/ml diluted in normal saline; Sigma) 90 minutes prior to sacrifice to label cells in S-phase. Sections were deparaffinized, rehydrated, incubated in 1% hydrogen peroxide for 15 minutes, immersed in Antigen Decloaker (Biocare Medical) and heated in a pressure cooker for 45 minutes. Sections were then blocked with Protein Block (Dako, Carpinteria, CA) for 10 minutes, and incubated with rat monoclonal anti-BrdU overnight at 4°C (1∶500; Accurate Chemical & Scientific). The following day, sections were incubated with goat anti-rat biotinylated secondary antibody (1∶500; Accurate Chemical and Scientific) for 1 hour, followed by HRP labeled Streptavidin (Accurate Chemical and Scientific) for 1 hour and then developed with diaminobenzidine and counterstained with hematoxylin. BrdU-stained cells were quantified in 100 well-oriented contiguous crypts.

Assessment of Def-GaPP Chitogel treatment efficacy on wound healing was determined by both macroscopic and histological analysis of wound healing, as described previously [30 (link)]. Wound samples were fixed in 10% neutral buffered formalin, embedded in paraffin wax, and cut at 4 µm thickness. Sections were then hematoxylin and eosin (H&E) stained, and wound length, dermal gape and re-epithelialisation were measured using the ImageProPlus program (Media Cybernetics Inc., Bethesda, MD, USA). Additional cut wound sections were processed for immunohistochemistry using previously established methods [30 (link)]. Sections were then deparaffinised and underwent antigen retrieval using target retrieval solution (TRS) in an Antigen Decloaker (Biocare Medical, Pacheco, CA, USA) at 90 °C for 10 min. Next, sections were blocked in 3% normal goat serum (NGS) for 30 min. Primary antibodies (Table S1) were then applied in 3% NGS in PBS overnight at 4 °C. After incubation, secondary antibodies (Table S2) were applied for 1 h at room temperature, then nuclei stained with DAPI (Sigma Aldrich, Sydney, Australia) at a 1:5000 (1 mg/mL stock). All sections were imaged using an Olympus IX83 Fluorescence Microscope (Olympus, Tokyo, Japan). Quantification of immunohistochemical staining included assessment of total positive cell numbers within the wound bed using the ImageProPlus 7.0 program.

Double stain IHC was performed for comparison of Injury-like and nmSC Core markers as follows. FFPE blocks from patient tumors were obtained from the Washington University Department of Pathology and were sectioned onto slides at 5 μm. Slides were baked at 60 degrees Celsius for 30 min followed by deparaffinization with xylene and graded ethanol. Antigen Decloaker (Biocare Medical) was used for heat-mediated antigen retrieval for all stains. Blocking was performed with Dual Endogenous Enzyme Block (DEEB, Agilent Dako) for 5 min. The first antibody was applied and incubated for 1 h. First antibodies included MHC II (1:400 dilution, Cell Signaling Technologies, clone LGII-612.4, catalog # 68258), Ngfr (1:100, abcam, clone NGFR/1965, catalog # ab224651), and S100 (1:25, Invitrogen, clone PA1-26313, catalog # PA1-26313). Sections were incubated with HRP Labeled Polymer (Dako) for 30 min followed by DAB staining for 5 min. Blocking was then repeated with DEEB. The second antibody was incubated for 1 h, then 30 min with Rabbit Polymer AP (Dako), and lastly AP Blue substrate for 15 min. Second antibodies included Sox10 (1:100, Cell Signaling Technology, clone E6B6I, catalog # 69661), SMARCC1 (1:800, Cell Signaling Technology, clone D7F8S, catalog # 11956), and CTCF (Cell Signaling Technology, clone D31H2, catalog #3418).