Figure 2. No label-retaining stem cells in the small intestinal crypt.

1. ¹⁵N-thymidine administered from post-natal day 4 - week 8. After 4-wk chase, BrdU was administered (500 µg i.p. Every 6 h) for 24 hrs before sacrifice (See Supplemental Fig 8).
2. ¹⁴N: crypt structure and intense signal in intracytoplasmic Paneth granules at the crypt base. ³¹P: intense intranuclear signal. ³²S: intense signal within cytoplasmic Paneth cell granules. ⁸¹Br: direct measure of BrdU incorporation. ¹⁵N/¹⁴N HSI: ¹⁵N-thymidine labeling within ¹⁵N+/BrdU- Paneth cells (large arrow) and mesenchymal cells (small arrows). No other cells reveal ¹⁵N retention. Large arrow head: recently formed (¹⁵N⁻/BrdU⁺) Paneth cell. Small hatched arrow (middle left side of the crypt): unlabeled Paneth cell, (¹⁵N⁻/Br-). Scale bar=15µm.
3. Continued analysis of the same crypt after narrowing the acquisition field. High ¹⁵N-signal in a BrdU⁻ Paneth cell (large arrow) and mesenchymal cells closely associated with the crypt (small arrows). BrdU⁺ CBC (hatched arrow) and Paneth cell (arrow head) are ¹⁵N⁻. Scale bar=5µm.
4. ¹⁵N/¹⁴N HSI image of small intestinal crypt at the end of ¹⁵N-thymidine pulse. All nuclei are labeled. Nuclei with lesser degrees of labeling likely represent cells born during a period of lower circulating ¹⁵N-thymidine as expected given the different labeling protocols (Supplemental Fig 8). Scale bar=20µm.
5. Unlabeled mouse image. The entire crypt contains the natural abundance of ¹⁵N. Scale bar=10µm.
6. Mean % ¹⁵N⁺ cells at the completion of pulse and after pulse-chase (± standard error of CBC, TA, Paneth, and mesenchymal (Mes) cells (n=3 mice per group).