TITLE: Immunofluorescent methods for antibody test in mouse colon
AUTHORS: Lixin Wang, Pu-Qing Yuan, Honghui Liang, Yvette Tache

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A table listed the information of antibodies used in the mouse colon. The protocol described the methods of colon tissues sampling, whole mount preparations, passive clarity technique, and respective fluorescent Immunohistochemistry for the antibody tests in different tissue preparations. </div></div>

[STEPS]
?. [Tissue Collection]
Mice were euthanized by an overdose of 5% isoflurane in oxygen.
?. [Tissue Collection]
After thoracotomy, perfusion cannula (18G) was inserted into the aorta via the left ventricle.
?. [Tissue Collection]
Saline was perfused for ~1 min to flush out the blood.
?. [Tissue Collection]
The colon was removed from ileocecal junction to the end of distal colon at the level of pelvic brim where runs the iliac artery, and flat-pinned on a Sylgard™ 184 silicone elastomer (Electron Microscopy Science, Hatfield, PA).
?. [Tissue Collection]
The colon was fixed in 4% paraformaldehyde at 4°C overnight.
?. [Tissue Collection]
The tissues were rinsed in 0.01 M phosphate-buffer saline (PBS).
?. [Whole Mount Preparation]
Whole mounts of the submucosal and myenteric plexuses of mouse proximal and distal colon were prepared under a stereomicroscope:Scrape off the mucosa using an angled fine tip forceps
?. [Whole Mount Preparation]
Peel off the submucosa layer containing the submucosal plexus using a micro-scissors.
?. [Whole Mount Preparation]
Remove the circular muscles carefully using fine forceps
?. [Whole Mount Preparation]
The remaining layers of the whole mount preparation includes the myenteric plexus adhered on the longitudinal muscle and serosa.Some samples had the circular muscles remained, i.e. the layers include the circular and longitudinal muscles with the myenteric plexus in between, and serosa, which are indicated in the antibody table.
?. [PACT (passive clarity technique)]
Rinse 3 times the colon samples remained flat-pinned on the Sylgard silicone dish in PBS with 0.02% NaN3
?. [PACT (passive clarity technique)]
Immerse the colon wall in Hydrogel A4P0 (4% acrylamide without paraformaldehyde) solution for 1 day at 4°C
?. [PACT (passive clarity technique)]
Place the colon in 50 ml tube with 5-10 ml hydrogel
?. [PACT (passive clarity technique)]
De-gas for 10 min in a desiccation chamber filled with nitrogen with a vacuum pump on (http://wiki.claritytechniques.org/index.php/CLARITY_Technique)
?. [PACT (passive clarity technique)]
Polymerize in 37°C water bath 1 h, then remove the extra gel as much as possible
?. [PACT (passive clarity technique)]
Rinse by 8% SDS overnight and then 4% SDS for 2 days in 37°C shaker
?. [PACT (passive clarity technique)]
Rinse 3 times in PBST (0.1% Triton-X in 1X PBS with 0.02% NaN3) at 37°C on a shaker plate 1 day, store in PBS-NaN3
?. [Fluorescent Immunohistochemistry]
For whole mount preparation and transverse sections:10% NDS in 0.3% Triton-X 100-PBS 1 h at room temperature (RT)
?. [Fluorescent Immunohistochemistry]
1st antibody in 0.3% Triton-X 100-PBS 2 h RT, 4°C 2 days
?. [Fluorescent Immunohistochemistry]
Rinse in 1X PBS 30’ x 5 times
?. [Fluorescent Immunohistochemistry]
2nd antibody RT 3 h
?. [Fluorescent Immunohistochemistry]
Rinse 1X PBS 5 times
?. [Fluorescent Immunohistochemistry]
Mount and coverslip in Vectashield (Vector Laboratories, Burlingame CA)
?. [Fluorescent Immunohistochemistry]
For FACT-cleared colon wall:10% NDS in 0.3% Triton-X 100-PBS 1 h RTat room RT, and then 4°C overnight
?. [Fluorescent Immunohistochemistry]
1st antibody in 0.3% Triton-X 100-PBS 3 h RT, 4°C 3-5 days
?. [Fluorescent Immunohistochemistry]
Rinse in 1X PBS 1 h x 5 times, then 4°C overnight
?. [Fluorescent Immunohistochemistry]
2nd antibody in 0.3% Triton-X 100-PBS 3 h RT, 4°C 1-2 days
?. [Fluorescent Immunohistochemistry]
Rinse in 1X PBS 1 h x 5 times, then 4°C overnight
?. [Fluorescent Immunohistochemistry]
Immersed in a custom-made imaging media, RIMS (ref) with a reflective index of 1.46], 2 h at RT and overnight at 4°C.
?. [Fluorescent Immunohistochemistry]
Embedded on microscopic glass slides in fresh RIMS sealed by iSpacer (SunJin Lab Co., Hsinchun City, Taiwan, www.sunjinlab.com). The colon samples were placed onto the slides with serosa on top.