Df6919

Immunofluorescence staining was performed using paraffin-embedded tissues as mentioned before. For immunofluorescence, the paraffin slides were deparaffinized, rehydrated, blocked and treated according to a standard protocol. The expression of intestinal barrier associated tight junction protein, P2Y₁₄R and EpCAM was evaluated by probing the tissues with primary antibody against Claudin-1 (bioss, bs-10011R, 1:300), Occludin (bioss, DF6919, 1:300), ZO-1 (Affinity, AF5145, 1:300), EpCAM (AiFang, AF04654, 1:300), Caspase-3 p-17 (Cell Signaling Technology, #9664, 1:1000) and P2Y₁₄R (Invitrogen, PAS-103202, 1:300) overnight at 4°C followed by 1 h incubation with the corresponding secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) (ab150114, Abcam, 1:500) or Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077, Abcam, 1:300). All slides were incubated with DAPI (S2110, Solarbio) for 10 min to show the location of the nucleus. Images were captured with a confocal microscope (LSM 800, Zeiss).

Intestinal samples were fixed overnight in 4% Carnoy’s fixative. Samples were then dehydrated in gradient ethanol (100%–95%−90%–75%−50%) and Xylene solution for at least 4 h. Then 4-µm paraffin sections were rehydrated, blocked with 3% BSA solution, and stained with antibodies overnight as follows: ZO-1 (Proteintech, 21773-AP, 1:2,000), E-cadherin (Affinity, AF0131, 1:200), and claudin-1 (Affinity, DF6919, 1:200). Tissue sections were then incubated with the appropriate fluorophore-conjugated secondary antibody. Before imaging, nuclei were counterstained with DAPI or Hoechst. Fluorescence analysis was performed on a Leica SP8 Fluorescence microscope.