An Immunofluorescence-Based Method for Visualizing Tuft Cells in Jejunum Cryosections

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.