Biodewax and clear solution

The chemical materials used in this study include SPRC (also named ZYZ‐802, Shanghai), carbomer 940 (Macklin, China), sodium carboxymethyl cellulose (Macklin), glycerin (Macklin), sodium hydroxide (Macklin), Tween‐20 (Sigma, USA), Tween‐80 (Macklin), TRIzol reagent (Carlsbad, CA, USA), and phosphate‐buffered saline (PBS) tablets (Gaithersburg, MD, USA). Recombinant bovine bFGF was purchased from Essex Bio‐Technology (Zhuhai, China). Hydrogen peroxide solution was obtained from ANNJET (Shandong, China). Alcohol, n‐butanol, and xylene were purchased from SINOPHARM (Shanghai, China). Masson Tricolor Staining Kit, HE Staining Kit, BioDewax and clear solution, tris‐ethylene diamine tetraacetic acid antigen retrieval solution (Tris‐EDTA), pH = 9.0), bovine serum albumin (BSA), horseradish peroxidase (HRP) conjugated goat anti‐rabbit IgG (H+L), and DAB chromogenic kit were purchased from Servicebio (Wuhan, China). Total RNA was extracted with RNA Extraction Kit was purchased from Solarbio (Beijing, China). The HiFiScript gDNA Removal cDNA Synthesis Kit was obtained from CWBIO (Jiangsu, China). The PerfectStar Green qPCR SuperMix (+Dye I/+Dye II) was purchased from TRANS (Beijing, China).

Conforming to the standard protocol, immunofluorescence staining was executed using the following antibodies and dilutions: TNF-α (1:500; Servicebio, Wuhan, Hubei, China), IL-6 (1:500; Servicebio, Wuhan, Hubei, China), and F4/80 (1: 1,000; Servicebio, Wuhan, Hubei, China). After being deparaffinized and rehydrated, the samples were subjected to epitope retrieval by microwaving the slides in an EDTA antigen retrieval buffer with a pH value of 8.0 (Servicebio, Wuhan, Hubei, China). The samples were first permeabilized with BioDewax and Clear Solution (Servicebio, Wuhan, China) and blocked with 3% BSA. Only one antigen was found in each stage, which included primary and secondary antibody incubation, as well as tyramine signal amplification (TSA) visualization. After protein blocking and epitope retrieval as mentioned prior, the next antibody was labeled. To finally stain the nuclei of the cells, the samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The immunofluorescent images were visualized and captured using the PANNORAMIC MIDI (3DHISTECH™, Budapest, Hungary) at 200× magnification. Semi-quantitative image analysis was performed with ImageJ software. The data were presented as relative fluorescence intensity to the Con group.

Paraffin-coated tissues were heated at 60 °C for 4 h and then deparaffinized with BioDewax and Clear Solution (ServiceBio). After deparaffinization, heat-induced antigen retrieval was performed. The tissues were subsequently blocked with 3% BSA at room temperature for 30 min. They were then incubated with the primary antibody overnight at 4 °C. The next day, the slides were treated with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature and subsequently exposed to DAB. Slides were then stained with hematoxylin, followed by decolorization using ethanol and hydrochloric acid, and finally fixed and dried. Images were captured under a microscope.