The overall goal of this staining procedure is to visualize tuft cells in mouse jejunum cryosections. This method can help answer key questions in the gastroenterology field, such as how the proliferation of tuft cells takes place in mammalian gut during parasite infection. The main advantage of this technique is that researchers with limited histrological experience can obtain high quality tuft cell images.
Begin by exposing the pelvic organs of a freshly formalin-fixed mouse cadaver. Cut off the end of the rectum from the anus, and separate the intestines from the body by cutting the mesentery. To isolate the jejunum, cut the intestines four centimeters from the anal side of the gastric antrum, and discard the anal half of the remaining small intestine.
Clip the jejunum in half to facilitate the flushing procedure. Load 20 milliliters of 10%buffered neutral formalin solution into a 20 milliliter syringe, and attach an 18-gauge straight needle with the bevel removed. Then, inject 10%buffered neutral formalin solution from one end of the clipped jejunum to flush out the intestinal contents, and to fix the gut lumen surface.
Wash the gut lumen by injecting PBS. Then, flush with liquid gelatin solution to replace the PBS. Close one end of the clipped jejunum by suture ligation, using a 6-0 nylon cut suture.
Fill the jejunum with liquid gelatin and close the opposite end by suture ligation. Next, tie four suture knots over the length of the jejunum. Soak the tissues in 50 milliliters of 15%sucrose in 10%buffered neutral formalin solution overnight, at 4 degrees Celsius.
The next day, cut the jejunum at the suture knots to obtain three sausage-like jejunem pieces with both ends ligated. Align the pieces in a cryomold, then cover with embedding compound. Snap freeze the cryomold in isopentane cooled with liquid nitrogen.
To prepare sections of jejunum, start by setting both the cryostat chamber temperature and object temperature to minus 22 degrees Celsius. Place the frozen tissue block in a cryomold in the cryostat chamber for at least 15 minutes. After 15 minutes, cut the block in half with a razor blade to expose the transverse section of the jejunum.
Mount one half of the block on a cryostat adaptor. Section the gelatin-filled jejunum into 30 micron thick sections. Use frozen forceps to gently transfer the sections into a 35 millimeter culture dish containing 3 milliliters of PBS.
After washing the sections three times for five minutes each with 3 milliliters of PBS-T, add 3 milliliters of freshly-prepared antigen retrieval solution to the dish containing the free-floating sections. Then, close the lid, seal the gap between the dish and the lid with a strip of vinyl tape, and incubate at 50 degrees Celsius in a hybridization incubator for three hours, without shaking. After immuno-staining, transfer the dish of stained sections in PBS to a stereoscopic microscope.
Place 200 microliters of PBS in a droplet on the center of a MAS-coded white glass slide. Then, use a P200 pipette tip to transfer one jejunum section from the dish into the droplet. After adjusting the section alignment, aspirate all remaining PBS surrounding the section.
Add 20 microliters of aqueous mounting media, and place a cover slip atop the media. Immediately seal the cover slip edges with xylene-based mounting media, and allow to dry for two to three hours before beginning confocal microscopy. Gelatin filling of the jejunum preserves the round disc shape and maintains upright positioning of the villi.
In the absence of gelatin filling, jejunal sections tend to kink, allowing the villi to swing backward. pY1798 reproducibly delineates entire tuft cells, including the membrane, cytoplasma of the spool-shaped soma, and the strongly stained luminal tip, where robust signal condensation corresponds to the protruding tuft of a tuft cell. Phalloidin has high affinity for filamentous actin, which is present in microvilli that form the intestinal brush border.
Phalloidin reproducibly and prominently marks the thickened brush border that corresponds to a mass of rootlets extending from the tuft. The consistent co-localization of pY1798 signals with the prominently thickened, phalloidin-positive brush border demonstrates that this protocol successfully identifies tuft cells regardless of whether they are located on a villus or in a crypt. Once mastered, this technique can be done in three days if it is performed properly.
The combination of low-emitting point gelatin, free-floating cryo-sections, and low-temperature antigen retrieval can be applied for immuno-staining other project tissues.
