Nbp2 25208

Paraffin sections were dewaxed sequentially in the order of 15 min for xylen I, 15 min for xylen II, 5 min for absolute ethanol I, 5 min for absolute ethanol II, 5 min for 90% alcohol, 5 min for 80% alcohol, and 5 min for 70% alcohol. Finally, the sections were washed with distilled water. After the sections were repaired with EDTA antigen recovery solution, they were incubated in 5% BSA for 30 min at room temperature. Gently shaked off the blocking solution, added anti-CD163 (ab182422, Abcam, 1:100), anti-CD206 (sc-58,986, Santa Cruz, 1:50), anti-CD86 (NBP2-25,208, Novus, 1:100), anti-HLA-DR (MA5-32,232, Invitrogen, 1:100). Then incubated overnight in a wet box at 4°C. The slices were placed in PBS (pH 7.4) and washed with decolorizing shaker for 3 times, 5 min each time. After the slices were slightly dried, the secondary antibody (dilution ratio: 1:200) corresponding to the primary antibody was dropped into the circle to cover the tissue, and incubated in the dark at room temperature for 50 min. DAPI counter-stained nuclei: the slices were placed in PBS (pH 7.4) and washed with decolorizing shaker for 3 times, 5 min each time. After the slices were shaken dried, DAPI dye solution was added dropwise to the circle, and incubated in the dark for 10 min at room temperature. Sealed the film and took pictures under the microscope.

All specimens were decalcified in 10% disodium Ethylenediamine tetraacetic acid (EDTA‐Na2, Solarbio, Beijing, China) at room temperature for ≈30 days. Subsequently, the decalcified samples were longitudinally embedded in paraffin wax and cut into 5 µm sections.
In order to detect phenotype switching of macrophages in vivo, immunofluorescence staining was performed. Tissue sections of 4 and 7 days were incubated with primary antibodies of anti‐CD86 (2 µg mL^−1, NBP2‐25208, Novus) and anti‐CD206 (10 µg mL⁻¹, ab64693, Abcam) at 4 °C overnight followed by incubation with the corresponding fluorescently labeled secondary antibody (CD86: CoraLite594‐conjugated Goat Anti‐Mouse IgG, CD206: CoraLite488‐conjugated Goat Anti‐Rabbit IgG; 1:200; Proteintech) and DAPI. The fluorescent images were obtained with a fluorescence microscope (Olympus), Image J was used to quantify the fluorescence intensity of the stained markers and normalized to DAPI‐stained nuclei counts.