Paraffin-embedded tissue blocks were cut into 2.5-μm slices and were mounted on polylysine-charged glass slides. Endogenous peroxidase activity was blocked by exposure to 3.0% H2O2 for 30 min. Antigen retrieval was performed in a citrate buffer (pH 6.0) at 120°C for 10 min. Sections were then incubated at 4°C overnight with anti-Brdu mAb (#B8434, 1:500 per mouse, Sigma-Aldrich). After washing, the sections were incubated with the corresponding secondary antibodies for 2 hours at room temperature. The VECTASTAIN ABC Kit (Vector Laboratories, San Diego, CA, USA) was used to perform the avidin-biotin complex method according to the manufacturer’s instructions. Sections incubated with isotype- and concentration-matched immunoglobulins without primary antibodies were used as isotype controls. Peroxidase activity was visualized with the DAB Elite Kit (K3465, DAKO), and brown coloration of tissues represented positive staining. A similar protocol was used to detect the expression of PCNA in colons.

To detect MS4A6D and VSIG4 colocalization, PEMs were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin in PBS for 5 min, and blocked with PBS containing 2% bovine serum albumin for 1 hour at 4°C. The cells were then stained with rabbit anti-MS4A6D antibodies and mouse anti-VSIG4 overnight at 4°C and then stained with Alexa Fluor 488–conjugated donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (#A-21206, Thermo Fisher Scientific, Billerica, MA, USA) as well as Alexa Fluor 555–conjugated goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (#A-21422, Thermo Fisher Scientific) for 1 hour. A similar protocol was used to detect the colocalization of IL-1β and F4/80 in colons. The results were analyzed using fluorescence microscopy (Zeiss Axioplan 2).

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