Characteristics of immune cell infiltration in inflamed mucosa of ulcerative colitis patients, hub gene candidates and key pathways in intestinal macrophage.

Tissue RNA was isolated from fresh mouse colon sample by Trizol reagent. Total RNA (1 µg) was reverse-transcribed using a reverse transcriptase kit (TakaRa, cat. RR036A-1, Tokyo, Japan). Hieff® qPCR SYBR Green Master Mix (Yeasen Biotechnolog, cat. 11201ES03, Shanghai, China) was used for Real-time qPCR. The comparative cycle threshold (Ct) method (2−ΔΔCT) was used to calculate relative expression, β-actin was used as the internal reference. We evaluated immunostaining by multiplying the intensity score and proportion score. Intensity score according to the intensity of cell staining: 0 for no positive staining (negative), 1 for light yellow (weakly positive), 2 for brown (positive) and 3 for dark brown (strongly positive). Proportion score according to the percentage of positive cells: 1 for ≤25%, 2 for 26%-50%, 3 for 51%-75% and 4 points for > 75%.