Immunofluorescence Staining of Tight Junctions

Immunofluorescence staining was performed [43 (link)] to evaluate ZO-1 and occludin expression in the colon. Anti-TSLP (rabbit, 1:50; GTX85059, GeneTex, Irvine, CA, USA), anti-IL-6R (rabbit, 1:1000; Invitrogen, Waltham, MA, USA), anti-ZO-1 (rabbit, 1:500; 61–7300, Invitrogen, Waltham, MA, USA), and anti-occludin (mouse, 1:200; OC3F10, Invitrogen, Waltham, MA, USA) antibodies were diluted in 1× PBST supplemented with 0.3% BSA. The slides were wrapped in an aluminum foil to block light and stored at 4 °C for 72 h. Tissue sections were incubated for 1 h with a mixture of Alexa 488-conjugated donkey anti-mouse secondary antibody (1:1000; A21206, Invitrogen, Waltham, MA, USA) or Alexa 594-conjugated donkey anti-mouse secondary antibody (1:1000; A21203, Invitrogen, Waltham, MA, USA).