Hrp labeled secondary antibody

BO (pharmaceutical batch number: 201671HZ) was supplied by the Department of Pharmacy at the Wuhan Integrated Traditional and Western Medicine Hospital (Wuhan, China). The BO used in this study is a hospital preparation which contains Rheum palmatum L., Angelica sinensis (Oliv.) Diels, Codonopsis pilosula (Franch.) Nannf., Asarum sieboldii Miq., Borneolum syntheticum, and Calomelas. Burn moisturizing scald ointment (BMS) was purchased from Meibao Pharmaceutical Co., Ltd. (Shantou, China); ELISA kits were obtained from Neobioscience Technology Co., Ltd. (Shenzhen, China); Collagen I primary antibody; phosphorylated PI3K, AKT, and mTOR; unphosphorylated PI3K, AKT, and mTOR polyclonal antibody; and HRP-labeled secondary antibody were purchased from Servicebio (Wuhan, China); Microscope (XSP-C204) was from Motic China group and microscope camera was purchased from Cognex (Massachusetts, United States); Toe volume measuring instrument (PV-200) was from TECHMEN Co., Ltd. (Chengdu, China); full-wavelength microplate reader 1,510 was obtained from Thermo Fisher Scientific (Massachusetts, United States); Ultimate 3000 series HPLC system consisting of the computer-controlled system with the CHROMELEONTM software and a SQL database, equipped with a WPS-3000 autosampler, was from Dionex (California, United States).

Tissue samples were fixed with 10% formalin, embedded in paraffin, and sectioned to an 8 μm thickness slide. Hematoxylin and eosin (HE) stain and Masson’s trichrome stain were then used to assess the extent of tissue fibrosis. In addition, heart slides were stained with Sirius Red and liver slides were stained with Oil Red to appraise hepatic steatosis. Images were captured using a Nikon Eclipse E100 ortho optical microscope (Nikon, Japan). For IHC, paraffin sections were dewaxed and rehydrated. Antigens were retrieved with citric acid antigen repair buffer and then put into 3% hydrogen peroxide solution to eliminate endogenous peroxidase. The slides were then blocked with 3% BSA and incubated with anti-TNF-α or F4/80 antibody (1:100) at 4 °C overnight. After incubating with HRP-labeled secondary antibody (1:1,000, Servicebio, Wuhan, China), the samples were detected using DAB staining. Images were acquired and analyzed using a Pannoramic 250 FLASH scanner (3D HISTECH, Budapest, Hungary).

Immunohistochemistry staining of paraffin sections was performed using a microwave-based antigen retrieval method. The heart tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were subsequently cut at 6-μm intervals perpendicular to the long axis of the heart. The primary antibody-rat CD47 (1:200, Abcam, Cambridge, MA, United States) was visualized using Alexa Fluor 550 secondary antibody (1:200, Servicebio, Wuhan, China). The primary antibody-rat CD68 (1:200, Abcam) for macrophages detection was visualized using HRP-labeled secondary antibody (1:200, Servicebio). The nuclei were stained with 4′, 6-diamidino-2-phenylindole dihydrochloride (Invitrogen, New York, United States). CD47/CD68-positive cells per square millimeter were counted under a 20× power field of the microscope in five random areas of LV tissues.