Immunostaining of Mucin-2 (Muc2) in Tissue Sections

Muc2 immunostaining was performed as previously described [25 (link), 50 (link)] with some modifications. Briefly, deparaffinized sections were incubated in 0.9M NaCl, 20mM Tris-HCl at pH7.2 and 0.1% SDS at 50°C for 3 hours, rinsed in PBS and blocked with 5% goat serum in PBS for 30 min at room temperature to minimize non-specific binding. Sections were then washed in PBS for 10 min prior to overnight incubation at 4°C with an anti-Muc2 rabbit polyclonal antibody (H300, Santa Cruz; 1:200 in PBS) [51 (link)]. Following incubation with primary antibody, tissues were washed 3 times in PBS for 10 min and incubated with goat-anti-rabbit Alexa 488 secondary antibody (Life Technologies, 1:1000 in PBS) for 1 hour at room temperature. Sections were washed twice in PBS for 10 min and counterstained with Hoechst (1:3000 in PBS). For FISH-Muc2 dual staining, sections were briefly rinsed in wash buffer after FISH hybridization and incubated directly with the anti-Muc2 primary antibody diluted in wash buffer. Incubation with secondary antibody was carried out at 4°C for 2 hours. A single 10 min PBS wash was performed after incubation with the primary and secondary antibodies before Hoechst nuclear staining and mounting with Mowiol solution.