An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway.

The goblet cell-associated antigen passages (GAPs), formed in the intestine and conjunctiva, contribute to immune responses by delivering antigens to immune cells. It has been proved that dysfunctions of GAPs may contribute to intestinal inflammation and dry eye disease. GAPs were first discovered in the intestine with the help of intravital two-photon microscopy. The imaging of GAPs was subsequently captured in a frozen section and whole-mount immunofluorescent staining. Both protocols have their features. This study provides a new protocol for visualizing GAPs with better mucus preservation by combining 4% PFA and methanol-Carnoy's solution (PFA-MC). In addition, embedding samples in paraffin provides an opportunity for antigen retrieval for immunohistochemistry analysis. Using this protocol, we have successfully visualized GAPs in the intestine, intestinal organoids, conjunctiva, and trachea. It is worth mentioning that the visualization of GAPs in intestinal organoids and airways has rarely been discussed before, which may assist researchers in their studies. Moreover, we also discovered the active uptake of dextran of club cells in terminal bronchioles, which may provide a new direction for studying the function of club cells. Overall, we developed a new method to visualize GAP formation and identify GCs simultaneously in the intestine, organoid, conjunctiva, and airway.