Ab46512

Distal colon tissue containing a fecal pellet was fixed by submersion in methanol-Carnoy solution for at least 24 h. Fixed tissue was paraffin embedded and cut into 5-µm sections. Tissue sections were deparaffinized by sequential washing in Xylene substitute (20 min at 60°C; Sigma-Aldrich) and 100% (5 min), 95% (5 min), 70% (5 min), and 30% (5 min) ethanol. Antigen retrieval was performed by immersion in 10 mM citrate buffer (95°C, 30 min). Sections were washed in PBS, permeabilized for 5 min using 0.1% vol/vol Triton X-100 (Sigma-Aldrich), and blocked using 5% vol/vol FCS. Sections were incubated overnight at 4°C with primary antibody solutions to stain Muc2 (Muc2C3, 1:500; Johansson et al., 2008 (link)), Apo-Muc2 (PH497, 1:1,000; Hansson et al., 1994 (link)), Clca1 (ab46512, 1:2,000; Abcam), Zg16 (anti-ZG16, 1:600; Rodríguez-Piñeiro et al., 2013 (link)), and Agr2 (H00010551-M01, 1:2,000; Abnova). Sections were washed in PBS, and primary antibodies were detected by incubation with goat anti-rabbit Alexa Fluor 488– or Alexa Fluor 555–conjugated secondary antibodies (1:2,000; Thermo Fisher) for 2 h at room temperature. Finally, slides were rinsed in double-distilled water and counterstained with Hoechst dye (5 µg/ml; Sigma-Aldrich) for 5 min. Stained slides were subsequently imaged using an LSM700 confocal microscope (Zeiss).