Anti ki67

Briefly, after deparaffinization and dehydration, specimens were boiled in 10 mM sodium citrate buffer to unmask antigens. Specimens were then blocked and incubated with primary antibody overnight at 4°C. Antibody binding was detected using Envision reagents (Boster bioengineering, Wuhan, PR China) according to the manufacturer's instructions. Primary antibodies used in this study were rabbit polyclonal anti-Ki67 (Epitomics, Burlingame, CA, USA, 1∶100 dilution), rabbit monoclonal anti-ERα (Epitomics, 1∶100 dilution), rabbit monoclonal anti-PTEN (Epitomics, 1∶100 dilution) and rabbit polyclonal anti-TIMP3 (Proteintech Group Inc., Chicago, IL, USA, 1∶50 dilution).

IHC staining was conducted as described previously [48 (link)]. Briefly, tissue sections were dewaxed and rehydrated before performing antigen retrieval. The slides were incubated with anti-ki-67 or anti-FLOT1 (Epitomics, Burlingame, USA) overnight at 4°C, and incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. DAB was used for color development, and dark brown staining was considered positive. The strength of positivity was semi-quantified by taking into account the staining intensity and the percentage of positive cells.

IHC staining was performed as previously described [42 (link)]. In short, tumor tissues from mice were dewaxed and rehydrated before conducting antigen retrieval. Slides were incubated with anti-HIF-1α (Cell Signaling Technology, Beverly, MA), anti-ki-67 or anti-active caspase-3 (Epitomics, Burlingame, USA) at 4°C overnight, followed by incubation with an HRP-conjugated secondary antibody at room temperature for 1 hour (h). Diaminobenzidine (DAB) was used for coloration, and dark brown was considered to be positive. The strength of positivity was quantified by considering the percentage of positive cells and the staining intensity.

For hematoxylin and eosin (H&E) staining, mouse colon tissues were fixed in 4% paraformaldehyde and embedded in paraffin and then incised a 4‐μm section. The sections were stained with both hematoxylin and eosin, and then photographed using an electron microscope.
For immunofluorescence staining, colon biopsies from UC patients and mice were embedded in O.C.T. compound (Tissue Tek, Sakura, Torrance, CA, USA), and then incised 8‐μm sections and processed for immunostaining. The DP1 expression was examined with polyclonal anti‐DP1 primary antibody (Cayman Chemical, Ann Arbor, MI, USA). The endothelial cells were marked with anti‐CD31 (1:200, BD Biosciences, San Diego, CA, USA). To detect epithelial cell and macrophage, anti‐pan‐keratin‐FITC antibody (Cell Signaling Technology, Danvers, MA, USA) and anti‐CD68 (1:200, AbD Serotec, Kidlington, UK) primary antibody were used. To label the smooth muscle cells, the anti‐α‐actin‐FITC antibody (1:200, Sigma‐Aldrich, St. Louis, MO, USA) was used. Anti‐CD301 (1:100 Bio‐Rad, California, USA) primary antibody was used to mark M2‐like macrophage, and anti‐Ki67 (1:500, Epitomics, Burlingame, CA, USA) primary antibody was used to label proliferating cell.

cSCC skin reconstruct were fixed with formalin for 2 h at room temperature, dehydrated and embedded in paraffin. The staining was performed using the UltraVision LP Detection System AP Polymer & Fast Red Chromogen assay (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, slides were treated with Ultra V Block and samples were incubated with anti-Ki-67 (1:200; Epitomics, Burlingame, CA, USA), or anti-keratin (ready to use; Cell Marque), or anti-MMP9 (1:100; Abcam, Cambridge, UK) or anti-psoriasin (1:50, Abcam, Cambridge, UK) for 1 h at room temperature.
After washes in PBS, Primary Antibody Enhancer (Thermo Fisher Scientific, Waltham, MA, USA) was added for 20 min at room temperature, followed by incubation with AP Polymer anti-mouse/rabbit IgG for 30 min at room temperature. Slides were stained with Fast Red using Naphthol Phosphate as substrate. Samples were analyzed under a conventional optical microscope (Zeiss Axioskope 40, Carl Zeiss, Jena, Germany).