Figure 3.

Characterization of Spermatogonia in the TOs by Immunofluorescent Staining of Whole Mounts

Immunostaining for UCHL1/DDX4 (first row), UTF1/DDX4 (second row), FGFR3/DDX4 (third row), and KI67/DDX4 (fourth row) in adult TOs (n = 3 TOs derived from different donors per staining) derived with (left and middle column) and without (right column) scaffold support following 24 hr (left column) and 4 weeks (middle and right column) of culture. Representative images show cells that stained positive for UCHL1, UTF1, or FGFR3 (green), and DDX4 (purple), which are unmistakably spermatogonia (white arrow). Cells positive for DDX4 alone represent a different germ-cell type (red arrow). The DDX4⁺ (purple) cells staining for KI67 (green) are dividing germ cells (white arrow). Cell nuclei are stained blue by DAPI. The inserts depict low-magnification overviews of the TOs generated using maximum intensity projection of z stacks. See also Figure S2 and Table S1.