Ab955

3-μm sections of paraffin-embedded kidneys underwent antigen retrieval by microwave heating in a solution of EnVision FLEX Target Retrieval Solution high-pH (Dako, K800421) for 20 min. Sections were incubated overnight at 4 °C with the following primary antibodies: CD68 (1:200, Abcam ab955), αSMA (1:400, Dako clone 1A4 M0851), FOLR2 (1:200, Invitrogen MA5-26933), TREM2 (1:100, R&D MAB17291) or collagen I (1:400, Invitrogen MA1-26771). Antigen detection was performed with alexa-fluor coupled antibodies: alexa fluor 488 goat anti mouse (for αSMA, Collagen I, CD68, FOLR2, Invitrogen A11001), alexa fluor-555 goat anti rabbit (for FAP, SFRP1, SFRP4 Invitrogen A21428), alexa fluor-647 goat anti rabbit (for FAP, SFRP4 Invitrogen A21245) or alexa fluor-555 goat anti rat (for TREM2 Invitrogen A21434). Nuclei were counterstained with Hoescht before mounting with anti-fade diamond mounting medium (Invitrogen P36970). Images were acquired by an upright widefield Apotome (Zeiss) or LSM 700 Zeiss confocal.

Colon sections were stained with hematoxylin and eosin (H&E) and graded for inflammation (0, none; 1, slight; 2, moderate; 3, severe), extent (0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural), crypt damage (0, none; 1, basal 1/3 damage; 2, basal 2/3 damage; 3, only surface epithelium lost; 4, entire crypt and epithelium lost), and percent of involvement (1, 1~25%; 2, 26~50%; 3, 51~75%; 4, 76~100%) as described^{37 (link)}. The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement to yield the final score. For evaluation using IHC, 5-μm-thick tissue sections were transferred onto adhesive slides and dried at 62 °C for 30 min. After incubation in 3% H₂O₂ to block the endogenous peroxidase activity, tissue sections were subjected to standard heat-mediated epitope retrieval in ethylene diamine tetraacetic acid (pH 8.0) for 32 min. The samples were incubated with primary antibodies against iNOS (1:50; PA1–036, Thermo Scientific, Rockford, IL), Arg-1 (1:100; PA5-29645, Thermo Scientific), and CD68 (1:2000; ab955, DAKO, Glostrup, Denmark), respectively. Immunostaining was detected using the DAKO IHC Detection Kit (K1492, DAKO). All IHC slides were counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene.