slides(Thermo Scientific), incubated for 10 min at 60%C and rehydrated through a series of graded alcohols and distilled water. Endogenous peroxidases were blocked by 3% hydrogen peroxide for 10 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6) by steaming sections in a microwave oven for 20 min. Tissue sections were blocked with 5% BSA/PBS for 30 min at RT and primary antibodies against MUC2(1: 200, sc15334, Santa Cruz Biotechnology) Ki67(1: 100, ab15580, Abcam) and Cleaved Caspase 3(1: 50, #9661, Cell Signaling Technology), were directly applied and incubated for I h at RT or overnight at 4C, espectively. Slides were washed 3x in PBs and secondary antibody (1: 200, UP511380 nterchim Optima) was applied for I hour at RT. Targeted antigens were visualized by using 3.3-diaminobenzidine solution (bd Pharmingen) followed by nuclear counterstain with hematoxylin. For immunofluorescence staining, staining procedure was done according to the protocol provided by Cell Signaling Technology. Primary antibody against MUC2 was used as indicated. Secondary antibody(1: 200, Al1008, Life Technologies) was applied for 1 hr at RT followed by nuclear counterstain with DAPI. Microscopic analyses were performed by using the Zeiss Axioplan 2 imaging microscope, Axio Imager ZI microscope, Axiovision software(all from Zeiss, Oberkochen, Germany), and ImageJ software(20). Evaluation of MUC2-and Ki67- positive signals was performed by counting MUC2-and Ki-67-positive epithelial cells in all intact villi and/or crypts per tissue sections. Caspase 3 cleavage was assessed by calculating ositively stained epithelial cells per mm mucousal area. Thickness of pre-epithelial mucus lyer in distal colon was obtained by calculating mean values of 10 distinct measurement points per tissue section Fluorescent in situ hybridization. Methacarn-fixed, paraffin-embedded colonic tissue sections (7 um) were deparaffinized in toluene, washed in 95 ethanol, and air-dried at RT, Tissue sections were incubated overnight(45C)in hybridization buffer(20 mM Tris-HCl, 0.9 M NaC 0. 1 SDS, pH 7.4) containing Cy3-labeled bacterial probes EUB338 (5 GCTGCCTCCCGTAGGAGT-3) and Bfra602 (5-GAGCCGCAAACTTTCACAA- espectively, in a concentration of 5 ng/ul(21-23). Non-specific binding of probes was removed by subsequent incubation of slides in pre-warmed hybridization buffer and washing buffer(20 mM Tris-HCl, 0.9 M NaCl, pH 7.4), both for 15 min at 37C. DAPI was used for nuclear counterstain and air-dried tissue sections were covered by using ProLongR Gold Antifade9 slides (Thermo Scientific), incubated for 10 min at 60° C and rehydrated through a series of graded alcohols and distilled water. Endogenous peroxidases were blocked by 3% hydrogen peroxide for 10 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6) by steaming sections in a microwave oven for 20 min. Tissue sections were blocked with 5% BSA/PBS for 30 min at RT and primary antibodies against MUC2 (1:200, sc15334, Santa Cruz Biotechnology) Ki67 (1:100, ab15580, Abcam) and Cleaved Caspase 3 (1:50, #9661, Cell Signaling Technology), were directly applied and incubated for 1 h at RT or overnight at 4°C, respectively. Slides were washed 3x in PBS and secondary antibody (1:200, UP511380, Interchim Uptima) was applied for 1 hour at RT. Targeted antigens were visualized by using 3.3’-diaminobenzidine solution (BD Pharmingen) followed by nuclear counterstain with hematoxylin. For immunofluorescence staining, staining procedure was done according to the protocol provided by Cell Signaling Technology. Primary antibody against MUC2 was used as indicated. Secondary antibody (1:200, A11008, Life Technologies) was applied for 1 hr at RT followed by nuclear counterstain with DAPI. Microscopic analyses were performed by using the Zeiss Axioplan 2 imaging microscope, Axio Imager Z1 microscope, Axiovision software (all from Zeiss, Oberkochen, Germany), and ImageJ software (20). Evaluation of MUC2- and Ki67- positive signals was performed by counting MUC2- and Ki-67-positive epithelial cells in all intact villi and/or crypts per tissue sections. Caspase 3 cleavage was assessed by calculating positively stained epithelial cells per mm2 mucousal area. Thickness of pre-epithelial mucus layer in distal colon was obtained by calculating mean values of 10 distinct measurement points per tissue section. Fluorescent in situ hybridization. Methacarn-fixed, paraffin-embedded colonic tissue sections (7 ȝm) were deparaffinized in toluene, washed in 95 % ethanol, and air-dried at RT. Tissue sections were incubated overnight (45°C) in hybridization buffer (20 mM Tris-HCl, 0.9 M NaCl, 0.1 % SDS, pH 7.4) containing Cy3-labeled bacterial probes EUB338 (5’- GCTGCCTCCCGTAGGAGT-3’) and Bfra602 (5’-GAGCCGCAAACTTTCACAA-3’), respectively, in a concentration of 5 ng/ȝl (21-23). Non-specific binding of probes was removed by subsequent incubation of slides in pre-warmed hybridization buffer and washing buffer (20 mM Tris-HCl, 0.9 M NaCl, pH 7.4), both for 15 min at 37°C. DAPI was used for nuclear counterstain and air-dried tissue sections were covered by using ProLongR Gold Antifade