Preparation and Immunohistochemistry of Mouse Small Intestine and Colon

Mice 6 - 8 weeks old were euthanized by isolfurane administration followed by cervical dislocation.

Mice were transcardially perfused with 5ml ice cold PBS.

Whole small intestine and colon were collected and kept in PBS on ice. The small intestine was dissected into three segments of approximately equal length.

Each segment (proximal, medial, and distal small intestine and whole colon) was flushed with 5ml ice cold PBS, cut lengthwise, and laid flat with the mucosal surface facing up. Each segment was further washed with ice cold PBS.

Tissue segments were washed with ice cold 4% PFA in PBS, and then laid flat in between two layers of filter paper and fixed in 4% PFA in PBS at 4C for either 4hrs or 24hrs.

Tissue was rinsed in excess PBS and transferred to 30% sucrose solution for at least 16 hours.

Tissue was transferred to a mixture of 50% OCT, 15% sucrose in PBS and rolled on a wooden stick.

Tissue was frozen in OCT as a "swiss roll" and sectioned into 5um or 20um sections on a cryostat and kept at -80C.

Slide-mounted tissue sections were thawed and air dried for 30 minutes

Sections were washed twice for 10 minutes in PBS and once for 10 minutes in .2% Triton X-100 in PBS.

Sections were blocked for 1 hour in blocking buffer at room temperature.

Sections were incubated with primary antibodies in blocking buffer overnight at 4C.

Sections were washed thrice for 10 minutes in .2% Triton X-100 in PBS.

Sections were incubated with secondary antibodies and DAPI in blocking buffer for 1-2 hours at room temperature.

Sections were washed twice for 10 minutes in .2% Triton X-100 in PBS and twice for 10 minutes in PBS.

Sections were mounted in Prolong Gold Mounting Media.