Whole Tissue Immunostaining of Mouse Colon

For whole tissue immunostaining, mouse colons were harvested and prepared as previously described^{34 (link)}. 5μm thick transverse sections were cut for hematoxylin and eosin staining and immunostaining. For this procedure, the sections were de paraffinized, hydrated, boiled in Trilogy solution (Cell Marque) for 20 minutes, rinsed in PBS, blocked with 10 mg ml⁻¹ bovine serum albumin/0.1% Triton-X100 for 30 minutes, and incubated with primary antibody at 4°C overnight. Primary antibodies include: goat anti-mouse pIgR (1/500, R&D Systems, catalog #AF2800) and goat anti-mouse IgA-AlexaFluor488 (1/200, Serotec, catalog #STAR137F). The slides were rinsed three times in PBS and incubated with AlexaFluor594-conjugated species-specific secondary antibody for one hour at room temperature (1/500, Invitrogen, catalog #A11058) if needed. Slides were washed three times in PBS and stained with bis-benzimide/Hoechst (Invitrogen) to visualize nuclei and mounted with a 1:1 PBS:glycerol solution. Staining was visualized with a Zeiss Axiovert 200 microscope with an Axiocam MRM digital camera.