Anti cd206

Frozen sections were incubated with anti-CD45 (1:200, BD), anti-Mac3-6 (1:50, Santa Cruz), anti-CD68 (1:200, AbD Serotec), anti-CD206 (1:200, Cell Signaling Technology), and anti-TFEB (1:200, Bethyl) antibodies, followed by incubation with secondary antibodies (1:300) (Thermo Fisher, Inc., Waltham, MA). The nuclei were counterstained with Hoechst 33342. The fluorescence signal was measured with a confocal laser scanning microscope (Leica, Germany).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA). BRB, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), HAuCl₄. 3H₂O (≥99.9% trace metals basis), BSA, Triton X-100 and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (USA). RIPA lysis buffer, phenylmethanesulfonyl fluoride, hematoxylin-eosin (HE) staining kit and Nissl staining solution (methylene blue) were obtained from Solarbio (China). The antibodies to anti-CD86, anti-CD206, anti-Cleaved Caspase-3 and anti-TNF-α were purchased from Cell Signaling Technology (USA). Anti-Bax, anti-Bcl-2, anti-IL-1β and anti-IL-6 were purchased from Affinity (USA). Anti-β-Tubulin, anti-GAPDH, HRP Affinipure Goat Anti-Mouse IgG and HRP Affinipure Goat Anti-Rabbit IgG were purchased from Proteintech (USA). The Alexa Fluor^®568 goat anti-mouse/rabbit IgG and Alexa Fluor^®488 goat anti-mouse/rabbit IgG were purchased from Invitrogen (USA). 4,6-dimethyl-2-phenylindole (DAPI) was purchased from Abcam (UK). RAW 264.7 cells and VSC 4.1 cells were obtained from the American type culture collection.

FFPE‐sectioned slides underwent rehydration to prepare samples for immunohistochemical staining and were subsequently incubated for 20 min in a citrate antigen retrieval buffer in a vegetable steamer. Following this, a gradual cooling process for 20 min occurred on the benchtop. The activity of endogenous peroxidases was quenched by a 5 min treatment in a solution of 0.3% hydrogen peroxide within 1x PBS. For staining, the VECTASTAIN Elite ABC‐HRP Kit (Vector Laboratories) was employed following the guidelines provided by the manufacturer (Vector Laboratories). Initially, after a wash in 1x PBS, the samples were blocked in 2.5% normal goat serum for one hour. Rabbit monoclonal anti‐CD11b (Abcam) and anti‐CD206 (Cell Signaling) were diluted at a 1:200 ratio in blocking buffer and incubated for one hour at room temperature. After three washes in 1x PBS, biotinylated secondary antibodies were introduced for a 30‐min incubation. Then, a 30‐min incubation with the VECTASTAIN Elite ABC Reagent (Vector Laboratories) was followed by three additional 1x PBS washes. ImmPACT DAB EqV Peroxidase Substrate (Vector Laboratories) was applied for either 1 min (CD206) or 30 s (CD11b). Slides were rinsed in tap water, subjected to a 5‐minute counterstaining with Harris Hematoxylin (Sigma), rinsed again, de‐stained with acid ethanol, dehydrated, and mounted with Permount (Fisher Scientific).