Hematoxylin and eosin

Frozen tumor sections were stained with hematoxylin and eosin (Wako Pure Chemical Industries Ltd.). Collagen was stained using a Masson’s trichrome staining kit (25088-100, Polysciences, Inc.). For immunochemical staining, frozen tumor sections were fixed in 10% formalin for 10 min at room temperature. After washing with 1 × TBS, sections were incubated with 0.3% H₂O₂ in methanol at room temperature, followed by washing in 1 × TBS. To inhibit non-specific staining, sections were incubated in serum-free protein block solution (X0909, Dako) for 15 min at room temperature. The sections were then incubated with primary antibodies: rat anti-mouse CD31 monoclonal antibody (BD557355, BD Biosciences, 1:100) or rat anti-mouse F4/80 monoclonal antibody (MCA497, Bio-Rad, 1:100). After washing with 1 × TBS, signal stain boost IHC detection reagent (anti-rabbit, Cell Signaling Technology) or rabbit anti-rat IgG H&L (horseradish peroxidase conjugated) (ab6734, Abcam, 1:500) was added, and sections were incubated at room temperature for 30 min. After reaction with diaminobenzidine (8059S, Cell Signaling Technology), sections were counterstained with hematoxylin. A BZ-X710 microscope (Keyence, Osaka, Japan) was used for histologic observation and evaluation.

After imaging with micro-CT, the skulls were isolated and fixed in 4% paraformaldehyde in PBS for 24 h at 4 °C and they were then dehydrated in a graded series of ethanol, embedded in paraffin, and sectioned at 7-mm intervals. After the sections were dewaxed and rehydrated, they were stained with hematoxylin and eosin (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

The skin tissues were collected and fixed overnight with 4% paraformaldehyde following by washing with PBS and soaking in 10% sucrose for 2 h and then 20% sucrose overnight. The wound tissues were embedded in O.C.T compound (Sakura Finetek, Tokyo, Japan) and frozen in liquid nitrogen to make frozen blocks of samples. The frozen wound tissue sections were mounted, stained with hematoxylin and eosin (Wako), and observed under a microscope (Olympus, Kyoto, Japan) to assess the tissue structure.
Immunohistochemical staining was used to analyze the role of AT-MSCs in recruiting inflammatory cells and forming vessels at wound sites. The inflammatory cells recruited to the ischemic area were examined by immunohistochemical staining of the wound tissues with rat anti-mouse CD45 (553078; BD Pharmigen Inc., San Diego, CA, USA), and rat anti-mouse Mac1 (rat anti-mouse CD11b 550282; BD Pharmigen). The neovascularization was analyzed by immunohistochemical staining of wound tissues with rat anti-mouse CD31 (553370; BD Pharmigen), according to the manufacturer’s instructions. The numbers of positive cells were counted in 10 randomly selected fields, and the average was determined.

The skin tissues were fixed overnight with 4% paraformaldehyde, then washed with PBS and soaked in 20% sucrose for 2 h. The wound tissues were embedded in O.C.T compound (Sakura Finetek, Tokyo, Japan) and frozen in liquid nitrogen. The frozen wound tissue sections on the seventh day post-injection were mounted, stained with hematoxylin and eosin (Wako, Osaka, Japan), and observed under a microscope (Olympus, Kyoto, Japan) to assess the tissue structure. Neovascularization was analyzed by immunohistochemical staining of wound tissues with rat anti-mouse CD31 (Lot. 53198; BD Pharmigen), according to the manufacturer’s instructions. The numbers of positive cells were counted in 10 randomly selected fields, and the average number was determined. For PKH26-labeled EV tracking, the tissue sections on the first day post-injection were mounted, stained with DAPI, and observed under a fluorescence microscope (Keyence, BZ-X710). The number of PKH26-positive signals was counted in 30 randomly selected sections per group, and the average number was determined.