This article details a protocol for immunostaining tuft cells in mouse jejunum cryosections. The method involves antigen retrieval, antibody application, and confocal microscopy to visualize cellular structures.
Begin with a culture dish containing formalin-fixed, gelatin-filled mouse jejunum cryosections featuring tuft cells with apical microvilli and spool-shaped soma.
Wash with a detergent-containing buffer to permeabilize the cell membranes.
Add an alkaline antigen retrieval solution. Incubate to break fixation-induced protein crosslinks, revealing the proteins for staining.
Apply a blocking solution to prevent non-specific antibody binding.
Overlay with primary antibodies targeting an actin-binding protein, specifically phosphorylated in tuft cells.
Remove unbound antibodies. Introduce a mix of fluorophore-conjugated secondary antibodies, DNA-binding dye, and phalloidin-fluorescent dye conjugate.
The secondary antibodies bind to primary antibodies, the phalloidin-fluorescent dye conjugate labels actin, and the DNA-binding dye marks cell nuclei.
Remove unbound components and add a detergent-free buffer.
Transfer the section to an adhesive-coated glass slide, mounting it with media.
Under a confocal microscope, green spool-shaped cells with intensified fluorescence at the luminal tip and an extended red rootlet mass indicate the presence of tuft cells.
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 cryo-mold 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 adapter. 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 5 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 immunostaining, transfer the dish of stained sections in PBS to a stereoscopic microscope. Place 200 microliters of PBS in a droplet on the center of an AMS-coated 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 coverslip atop the media. Immediately seal the coverslip edges with xylene-based mounting media, and allow to dry for two to three hours before beginning confocal microscopy.
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