Encyclopedia of Experiments: Immunology
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Transcript
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.
