Volume 115, Issue 4, Pages 898-908 (October 1998) Identification of neurons that express stem cell factor in the mouse small intestine  Heather M. Young, Shigeko Torihashi, Daniela Ciampoli, Kenton M. Sanders  Gastroenterology  Volume 115, Issue 4, Pages 898-908 (October 1998) DOI: 10.1016/S0016-5085(98)70262-8 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Fluorescence micrographs of whole-mount preparations of myenteric plexus with attached circular muscle after immunohistochemical processing. (A) NOS-immunoreactive cell bodies were abundant in the myenteric plexus, and NOS terminals were present in the myenteric plexus and in the overlying circular muscle (arrows) at P0 (bar = 50 μm). (B) At P0, calretinin-immunoreactive neurons were present in the myenteric plexus; the nuclei of these cells were strongly stained. Calretinin nerve terminals were sparse in the myenteric plexus and absent from the circular muscle (bar = 50 μm). (C and D) Paired micrographs showing the distribution of (C) calretinin and (D) choline acetyltransferase (ChAT) immunoreactivities in myenteric neurons of a P10 mouse. Calretinin-immunoreactive neurons (black asterisks) also contained ChAT (white asterisks; bar = 25 μm). (E and F) Paired fluorescence micrographs showing calretinin-immunoreactive nerve terminals (E) in the circular muscle of a P10 mouse, which were also immunoreactive to ChAT (F). Arrows indicate the same nerve terminals (bar = 10 μm). (G) Calbindin-immunoreactive cell bodies were abundant in the myenteric plexus at P0 (bar = 50 μm). (H) Confocal microscope image (projection of 20 optical sections, 0.5-μm z steps) of a calbindin-immunoreactive neuron and calbindin terminals in a myenteric ganglion of a P0 mouse. The neuron gave rise to three processes, and there were calbindin terminals present in the ganglion (arrows; bar = 25 μm). Gastroenterology 1998 115, 898-908DOI: (10.1016/S0016-5085(98)70262-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Overlay of fluorescence micrographs showing NOS cells (green) and calretinin cells (red) in the myenteric plexus from a P1 mouse. There was no overlap between NOS and calretinin (bar = 10 μm). (B) Overlay of a light micrograph of β-galactosidase staining (blue rims around nuclei) and a fluorescence micrograph of NOS immunoreactivity (green). Two of the cells (1) showed both β-galactosidase staining and NOS immunoreactivity, another cell (2) showed NOS immunoreactivity but no β-galactosidase staining, and the other labeled cell in the micrograph (3) showed β-galactosidase staining only (bar = 10 μm). (C and D) Paired micrographs showing the distribution of β-galactosidase staining (C) compared with that of calbindin immunoreactivity (D) in the myenteric plexus from a P0 mouse. Two of the cells in the field of view (asterisks) showed both β-galactosidase staining and immunoreactivity to calbindin, and there was also a cell that showed β-galactosidase staining only (arrow; bar = 20 μm). (E) Overlay of a light micrograph of β-galactosidase staining (blue) and a fluorescence micrograph of calretinin immunoreactivity (red) from a P0 animal. In this field of view, none of the β-galactosidase–stained cells (white asterisks) contained calretinin, and none of the calretinin-immunoreactive cells (black asterisks) showed β-galactosidase staining (bar = 10 μm). (F) Overlay of a light micrograph of β-galactosidase staining and fluorescence micrographs of NOS (green) and calbindin (red) immunoreactivities. Some of the cells (1) showed NOS immunoreactivity and β-galactosidase staining, some showed NOS staining only (2), one cell showed β-galactosidase staining only (3), and one cell showed calbindin and β-galactosidase staining (4) (bar = 10 μm). Gastroenterology 1998 115, 898-908DOI: (10.1016/S0016-5085(98)70262-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Percentages (means ± SEM) of different classes of enteric neurons that express SCF. The decrease in the percentage of each cell class that expresses SCF with age is consistent with the fact that the proportion of total myenteric neurons that expresses SCF decreases with age.12 Gastroenterology 1998 115, 898-908DOI: (10.1016/S0016-5085(98)70262-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Classes of SCF-expressing neurons at different ages. Gastroenterology 1998 115, 898-908DOI: (10.1016/S0016-5085(98)70262-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 (A and B) Paired micrographs showing (A) calretinin immunoreactivity and (B) the pattern of β-galactosidase staining in the myenteric plexus of a P1 mouse. One of the calretinin-immunoreactive cells (1) gave rise to many dendrites and a single axon (arrow). This cell also showed β-galactosidase staining. The other calretinin-positive cells (2, 3, and 4) were only weakly immunoreactive and did not appear to give rise to any processes; they also did not show β-galactosidase staining. Some of the β-galactosidase–stained cells (black asterisks) did not show immunoreactivity to calretinin (bar = 25 μm). (C) Another example of a calretinin cell (black asterisk) from a P1 mouse that showed both calretinin immunoreactivity and β-galactosidase staining (not illustrated). This cell possessed multiple dendrites and a single axon (arrow). The other three calretinin cells in the field of view (white asterisks) did not give rise to any processes and also did not show β-galactosidase staining (bar = 25 μm). (D) A calretinin immunoreactive axon that gave rise to many nerve terminals in a myenteric ganglion (bar = 25 μm). The images of the calretinin neurons in A-D are confocal microscope images (projection of 17-21 optical sections, 0.5-μm z steps). Gastroenterology 1998 115, 898-908DOI: (10.1016/S0016-5085(98)70262-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 β-Galactosidase staining in P21-P60 myenteric neurons. (A and B) Pattern of (A) NOS and (B) β-galactosidase staining in a P60 mouse. Only one of the NOS cells in the field of view (white asterisk) shows β-galactosidase staining. There is also a β-galactosidase–stained cell (black asterisk) that does not contain NOS. (C-E) Pattern of (C) ChAT, (D) calretinin, and (E) β-galactosidase staining in a group of myenteric neurons from a P21 mouse. The calretinin cells (1) all show ChAT immunoreactivity but no β-galactosidase staining. Of the two ChAT cells that do not show calretinin immunoreactivity (black asterisk, 2), only one cell (black asterisk) shows β-galactosidase staining. The reaction product in the β-galactosidase–stained neuron is mainly associated with the Golgi apparatus, as described by Torihashi et al.12 (F-H) A calbindin neuron (G) from a P21 mouse shows ChAT immunostaining (F) and β-galactosidase staining (H) (bars = 10 μm). Gastroenterology 1998 115, 898-908DOI: (10.1016/S0016-5085(98)70262-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions