G1211

Single label immunohistochemical assays were performed using primary antibodies for specific biomarkers (Table 1). Sample slides were incubated with the appropriate primary antibodies overnight at 4°C and subsequently labelled with secondary antibody (Caprice anti‐rabbit, G23303, 1:200 dilution, from Servicebio, China) and HRP at room temperature for 50 min (Servicebio: G1211). The slides were then dried, and freshly prepared DAB chromogenic reagents were used to label the marked tissue. Reaction times were monitored by microscopy until cell nuclei showed a brown‐yellow colour. Nuclei were counterstained with haematoxylin staining solution (G1004, Servicebio Cor, USA). The slides were then treated with the differentiation solution (G1039, Servicebio Cor, USA) for a few seconds and washed under running tap water. Then, the slides were treated with a bluing solution (G1040, Servicebio Cor, USA) and washed under running tap water. Positive cells as identified by DAB reagent (G1211, Servicebio Cor., USA) showed brown‐yellow nuclei.
Overall, 16 distinct single‐stained bio‐marker samples from 3 organs were evaluated by microscopy. Images were captured (Microscope Camera XSP‐C204, Olympus Europa GmbH, Hamburg, Germany) at a magnification of 400 × at 300 dpi in TIFF format. Photoshop software was used to false‐colour fluorescent markers in the images.

Immunohistochemical analysis was performed using anti-TLR4 (1:1000, GB11519, Servicebio), anti-MyD88 (1:200, GB11269, Servicebio), and anti-NF-κB p65 (1:400, bs-0465R, Bioss) antibodies. Fixed tissue gradient alcohol dehydration, paraffin embedding, and slicing to a thickness of 4 μm were performed. After deparaffinization and rehydration, the tissue sections were antigen-repaired with citric acid antigen-repair buffer (pH 6.0) and heated for the specified time. After natural cooling, the slides were placed in PBS (pH 7.4) and washed by shaking on a decolorization shaker three times for 5 min each. After treatment with 3% H₂O₂ and incubation at room temperature in the dark for 25 min, the slides were placed in PBS (pH 7.4) and washed by shaking on a decolorization shaker 3 times for 5 min each. Subsequently, 3% BSA was added to the circle to cover the tissue evenly, and the tissues were sealed for 30 min at room temperature. After gently removing the sealing solution, the sections were placed flat in a wet box with primary antibodies and incubated overnight at 4°C, followed by incubation with secondary antibodies for 30 min. The sections were then stained with diaminobenzidine (DAB) (G1211, Servicebio) and hematoxylin and mounted with neutral balsam. Brownish-yellow granules indicated positive signals. The slides were scanned and evaluated using Image-Pro Plus software.