G1340

The heart sections from four experimental groups were subjected to Masson's trichrome staining according to the protocol (G1340, Solarbio, China). The ratio of fibrosis was calculated as the ratio of fibrotic area to total heart area × 100 %.

The endometrial mouse tissues were fixed in 4% paraformaldehyde (Biosharp, BL539A, China) and subsequently embedded in paraffin. Following the kit instructions, tissue sections were cut into 5-μm thickness and stained with hematoxylin and eosin or Masson's Trichrome (Solarbio, G1340, China). The sections were then dewaxed by three immersions in histoclear for 10 min each before rehydration with 100%, 70%, and 50% IMS as well as dH₂O for 5 min each. Finally, the sections were incubated overnight at 4 °C with primary antibodies (diluted in antibody diluent reagent solution) The sections were subsequently incubated with HRP-conjugated secondary antibodies (1:1000, Abcam, ab150077, UK) at room temperature for 8 min. Afterward, DAB substrate (Solarbio, DA1010, China) was utilized to visualize the antigen signals and hematoxylin was used as a counterstain. The sections were then examined under a microscope (DMi8, Leica, Germany), and optical densities were quantified using Image-pro plus software (Media Cybernetics, USA). The list of antibodies employed in IHC is provided below: CDH2/N-cadherin (1:200, Abcam, ab18203, UK), CDH1/E-cadherin (1:400, Abcam, ab76055, UK), α-SMA (1:200, Abcam, ab7817, UK), Smad2/3 (1:200, ABclonal, A11498, China), VEGF (1:200, Abcam, ab52917, UK), IGF-1 (1:200, Abcam, ab9572, UK).

Expanded skin specimens were harvested during flap transfer surgery and fixed with formalin, embedded in paraffin, and sectioned at a thickness of 6 μm. Hematoxylin and eosin (HE) (G1120; Solarbio, Beijing, People’s Republic of China) staining and Masson trichrome (MT) (G1340; Solarbio) staining were performed for histologic examination. Differences in skin thickness were observed with HE staining in both groups, and collagen synthesis was evaluated using the collagen volume fraction (CVF) based on MT staining. Proliferating cell nuclear antigen (PCNA) (BM0104; Boster, Wuhan, People’s Republic of China) staining was performed to evaluate cell proliferation and CD31 (BA2966; Boster) staining for vascularization in skin sections of both groups.
The stained images were captured using a light microscope (Leica, Wetzlar, Germany) and Nikon DS-Ri2 digital camera with NIS Elements software (Nikon, Tokyo, Japan). The mean values of the thickness of epidermis and dermis, and CVF, count of PCNA-positive proliferating cells and CD31-positive blood vessels, were calculated independently in five random high-power fields (HPFs).