Three-dimensional high-resolution fluorescence microscopy shows Jacob lectins and chitinase are expressed early during encystation of Ei in vitro, while Jessie lectins are expressed late during encystation.

<p>Ei were allowed to encyst for 12 hrs (A to D), 24 hrs (E to H), 36 hrs (I to L), 48 hrs (M to P), or 72 hrs (Q to T) before they were fixed, permeabilized with non-ionic detergent, and then directly labeled with red anti-Jacob antibodies, green anti-chitinase antibodies, and blue anti-Jessie3 antibodies. At each time point, the same organism is shown with the three labels, and the merged three-color images are shown in the right hand-column. Not shown are control Ei trophozoites, which do not bind antibodies to Jacob, chitinase, or Jessie3. Also not shown are negative controls with non-immune rabbit antibodies, which did not bind to encysting organisms. Jacob lectins form large vesicles early during encystation; Jacob is the first lectin to appear on the surface of encysting Ei; and Jacob is a major component of the mature cyst wall. Chitinase appears next and is present in large vesicles that rarely overlap with those of Jacob lectins. In contrast to Jacob, most of the chitinase does not remain on the surface of mature cysts. Vesicles containing Jessie3 lectins appear late, and for the most part Jessie3 lectins are targeted to the cyst wall. Bar is 10 microns. Each image is a composite of multiple optical sections.