Haematoxylin eosin staining was applied for optical microscope observation. Controls were performed by replacing the primary antibody with buffer solution. The density of positive staining cells was calculated by MetaMorph®

Imaging System (Downingtown, PA, U.S.A.) After fixation in 4% paraformaldehyde, the specimen was kept in 30% sucrose at 4 °C overnight, and sectioned at 5 μm in thickness by freezing microtome. Then, it was incubated in 3% H2O2 at 37 °C for 30 min, rinsed in PBS buy Ipatasertib for 3 times, and blocked with 5%BSA at 37 °C for 30 min. Specimen was treated by chicken anti-Ag85A IgY (1:400) at 4 °C overnight, followed by rinsing in PBS for 3 times, and incubated with FITC-goat-anti-chicken find more IgY (1:200, Gene Corporation) at 37 °C for 30 min. It was provided for fluorescence microscopic observation after sealing samples with 10% glycerol. The density of positive staining cells was calculated by MetaMorph® Imaging System, as shown in total grey value average. The procedure is in the similar manner as described in Section 2.4, except replacement of the primary antibody with chicken anti-Ag85A IgY (1:400) at 4 °C

overnight. After intensively washing, they were incubated with TRITC conjugated UEA-1 (1:40, Vector Laboratories) and FITC-goat-anti-chicken IgY (1:200, Gene Corporation) simultaneously at 37 °C for 30 min, and provided for fluorescence microscope observation as described. The procedure is in the

similar manner as described in Section 2.4, except replacement of the primary antibody with chicken anti-Ag85A IgY (1:400) and purified Armenian Hamster-anti-mouse CD11c (1:20, BD Pharmingen Corporation) and secondly replacement of the antibody with Texas Red conjugated Goat Anti-Armenian Hamster IgG (1:75, Jackson ImmunoResearch Laboratories) and FITC-goat-anti-chicken IgY (1:200). Total RNA from 2 × 106 IELs was extracted by Rneasy Mini Kit (QIAGEN, China) according to the manufacturer’s instructions. DNA of FasL and β-actin (as internal parameter) was respectively amplified by PCR with sense primer5′-AAT TAC CCA TGT CCC CAG ATC-3’and that of antisense primer was 5′-GCT GCT GTG GGC CCA TAT CTG-3′ for FasL gene. For β-actin gene, sense primer was 5′-TCA GAA GGA CTC CTA TGT GG-3′ and that of antisense primer Edoxaban was 5′-TCT CTT TGA TGT CAC GCA CG-3′. The total volume of PCR system was 50 μl. PCR cycling conditions were; pre-denaturation at 95 °C for 2 min; denaturation at 95  °C for1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min, in total 35 cycles; and extension at 72 °C for 10 min. Size of FsaL product amplified was 709 bp and that of β-actin was 500 bp. The products were scanned and analyzed by ChemiImager 5500 gel imaging analyzer (UVP, USA) after electrophoresis and staining by Ethidium Bromide. FasL amount expression was measured as the density of FasL and β-actin. IELs were isolated as described [15].