Dab substrate kit

The cells were seeded on Nunc Lab-Tek chamber slides (Thermo Fisher Scientific) and incubated at 37°C until confluent. Cells were subsequently washed twice with PBS and fixed with 3.7% formalin at room temperature for 15 min. The cells were permeabilized with 0.25% triton in PBS for 10 min and blocked with 2% bovine serum albumin (Wako Pure Chemical Industries) in PBS for 1 h. Cells were incubated at room temperature for 2 h with HRP-labeled anti-PPAR-α antibody (1:200, Santa Cruz Biotechnology, catalog no. sc-398394). The cells were rinsed three times with PBS and then stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB) using a DAB substrate kit (Nichirei Biosciences, Tokyo, Japan). The cells were counterstained with Meyer's hematoxylin, dehydrated, cleared with 99% xylene for 15 min, and mounted in malinol (Muto Pure Chemicals, Tokyo, Japan). The expression of PPAR-α in cells was observed at 200x magnification using a light microscope (BX53; Olympus Corporation, Tokyo, Japan), and images were captured with a microscopic camera (DP20-5; Olympus Corporation).

Perilipin is a lipid droplet surface protein specifically expressed in adipocytes [7 (link)]. Perilipin immunostaining was performed to examine the localization of adipocytes in the FGF-2-impregnated bilayer artificial dermis. Deparaffinized sections were used for immunostaining of perilipin. Before the primary antibody was added, antigenic sites were treated with ethylenediaminetetraacetic acid (EDTA). The slides were immersed in 3% hydrogen peroxide solution for 10 min to inhibit endogenous peroxidase before incubating for 5 min in Tris-buffered saline-T (TBST) (50 mM Tris–HCl, pH 7.6, 0.15 M NaCl + 0.05% Tween). To detect perilipin expression, slides were then exposed to rabbit monoclonal anti-perilipin antibody (Cell Signaling Technology Japan, Tokyo, Japan), diluted 1:100 in phosphate-buffered saline (PBS) at 4 °C overnight. After washing in TBST, Simple Stain MAX-PO (R) (NICHIREI BIOSCIENCES INC, Tokyo, Japan) was added to the slides for 30 min at room temperature. After washing in TBST, the slides were exposed to the DAB-Substrate kit (NICHIREI BIOSCIENCES INC) according to the manufacturer’s protocol. The thickness of the newly formed adipose tissue and that of the part in which collagen remained were measured on the stained specimens from the center of the samples, and the values were compared between the FGF-2 and control groups.

The dorsal skins were fixed in 10% neutral buffered formalin for paraffin-embedded specimen and 4% paraformaldehyde for frozen specimen. Embedded tissues were sectioned at 3 μm (paraffin-embedded specimens) or 8 μm (frozen specimens). Paraffin-embedded specimens were stained with hematoxylin and eosin (H&E) and toluidine blue (TB). Epidermal thickness was measured in five randomly selected areas (900 × 700 μm) of each H&E-stained sample. Mast cells were counted in ten randomly selected areas (450 × 350 μm) of each TB-stained sample. For immunohistochemistry, the paraffin-embedded sections were deparaffinized using xylene and rehydrated with ethanol. Frozen specimens were dried sufficiently with a dryer. Antigens were activated using a Histofine simple stain kit (Nichirei Bio Sciences, Tokyo, Japan). The antibodies used are described in Supplementary Table 2. Signals were visualized with a DAB Substrate Kit (Nichirei Bio Sciences). Sections were counterstained with hematoxylin. p-ERK immunostained area/epidermis (%) was measured in five randomly selected areas (900 × 700 μm) of each p-ERK-stained sample.

Tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were stained with H&E. IHC was performed using anti-F4/80 (Abcam, ab6640), anti-Mac2 (Cedarlane, CL8942AP), anti–CSF-1R (Abcam, ab215441), anti-CD45R (BD Biosciences, 550286), anti-MPO (Abcam, ab9535), and anti-EPX (Biorbyt, orb5168) antibodies, and the antigen-antibody binding was detected using Simple Stain MAX PO (Nichirei) and the DAB Substrate Kit (Nichirei).