Dab color development kit

RPMI 1640(SH30809.01), TRYPSIN 0.25% Solution (SH30042.01), and PBS buffer (SH30256.01B) were purchased from HyClone; Penicillin-streptomycin solution (100 × , C0222), PE anti-mouse CD40(124609), APC anti-mouse CD86 (105011), PerCP/Cy5.5 anti-mouse CD80 (104721), and FITC anti-mouse CD11c(117305) were purchased from Beyotime; Murine M-CSF(315-02-10 μg) and IL-4(214-14-20 μg) were purchased from PEPROTECH; FBS (P30-3302) was purchased from PAN; Goat Anti-Mouse IgA alpha chain (HRP) (ab97235), Goat Anti-Mouse IgG1 heavy chain (HRP) (ab97240), and Goat Anti-Mouse IgG2a heavy chain (HRP) (ab97245) were purchased from Abcam; Mouse TNF-α ELISA kit (1217202), Mouse IL-12p70 Elisa kit (1211202), Mouse IL-10 ELISA kit (1211002), and Mouse IL-6 ELISA kit (1210602) were purchased from Dakewe; Lowry protein concentration assay kit (PC0030), DAB color development kit (DA1010), and KDO (2-keto-3-deoxyoctane, K2755) was purchased from Solarbio; Quant-iT™ PicoGreen® dsDNA Reagent (P7581) was purchased from Thermo (Invitrogen).

PC paraffin tissue sections (Shanghai Outdo Biotech (HPanA020PG02)) were dewaxed and rehydrated using xylene and gradient alcohol, respectively, and rinsed twice with PBS-T for 5 min each. Endogenous peroxidase was removed with 3% H₂O₂ (in methanol), and antigen repair was performed using EDTA solution (0.05 M, pH 8.0) at 85° C. Next, the tissue sections were cooled to room temperature, rinsed thrice with PBST for 5 min each, and blocked for 2 h with 3 mL of blocking solution (Sangon Biotech, China). The specimen was incubated with the decorin primary antibody (Abcam, Cambridge, MA, USA), followed by the HRP-labeled secondary antibody. The tissue sections were visualized using the DAB color development kit (Solarbio, Beijing, China), and counterstained with hematoxylin and eosin for 2 min, fractionated with HCl for 30 s, blued with tap water, and finally sealed with glycerin gelatin sealing tablets (Solarbio). Nikon E80i microscope (Nikon, Tokyo, Japan) was used to observe and photograph the sections and Image J was used to quantify the signal of decorin for each individual stage (I-III).