Ab208687

Considering that the BDDM is full of collagen, we used it as a collagen scaffold to evaluate the binding ability of CBD–bFGF. A reference for the collagen binding assay can be found in published literature (Shi et al., 2017 (link)). Briefly, the BDDM (weighing approximately 122.71 ± 1.87 mg) was plated in a 48-well plate and then incubated with gradients of equal molar amounts of NAT-bFGF or CBD–bFGF for 2 h at 37°C. A primary antibody (ab208687, anti-bFGF, Rabbit, Abcam, United States) was added to the plates for 1 h at 37°C. Unbound antibody was removed by three washes with PBS. A secondary antibody (ab97048, goat anti-rabbit IgG H&L, Abcam, United States) was then added, followed by incubation for 1 h at 37°C. After washing with PBS, P-NPP disodium (2 mg/ml) was added for 10 min at room temperature, followed by termination of the reaction by the addition of an equal volume of 0.2 M NaOH. The absorbance was measured at 405 nm using a plate reader.

The detailed protocol was described previously^{7 (link)}. In brief, cell sheets were transferred onto a CellShifter (CellSeed Inc., Tokyo, Japan) to prepare tissue sections. Isolated cutaneous tissues or cell sheets were fixed in a 10% formalin neutral buffer solution and embedded in paraffin. Three-micrometer-thick sections were mounted on slides and stained with hematoxylin–eosin (HE) or Azan. Immunostaining was performed using primary rabbit antibodies for anti-collagen I (1:200, ab34710, Abcam, Cambridge, UK), anti-FGF-2 (1:30, ab208687, Abcam), and anti-HMGB-1 (1:400, ab79823, Abcam), rabbit anti-CD31 antibody (1:200, ab28364, Abcam) and a secondary antibody for DyLight550 conjugated goat anti-rabbit IgG (H&L) (1:200, ab96884; Abcam). DAPI was used to stain the cell nuclei. All histological images were captured by BZ-X710 microscope and analyzed using BZ-X analyzer (Keyence). All histological analyses were performed by a pathologist.

Proteins were isolated from cells using RIPA lysis buffer (Thermo Scientific) and quantified with a BCA assay kit (Bio‐Rad, Hercules, CA, USA). Equal amounts of protein samples (20 μg) were dissolved by 10% SDS-PAGE (Bio-Rad) and blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Beijing, China). Membranes were blocked with 5% defatted milk and incubated at 4°C overnight with primary antibodies against fibroblast growth factor 2 (FGF2, ab208687), vascular endothelial growth factor (VEGF, ab46154), angiopoietin 1 (Ang1, ab183701), β-actin (ab115777) (all from Abcam, Cambridge, MA, USA) and GMFG (13625-1-AP; Proteintech, Chicago, IL, USA), followed by incubation with the secondary antibody (ab7090, Abcam) at room temperature for 2 h. The blots were visualized with the ECL kit (Vazyme, Nanjing, China) and quantified with ImageJ software (GE Healthcare).