**2.5 Immunostaining of Fem1b in pancreatic islets**

In humans, FEM1B has been shown to be expressed within whole pancreas [10], but cell type distribution within this organ was unknown. Immunostaining of wild-type pancreas with immunoaffinity-purified antibody shows that Fem1b protein is expressed in pancreatic islets (Figure 5).

Fig. 5. Lower and higher magnification, respectively, of immunoperoxidase staining with antibody Li-51 (against the C terminus of Fem1b) counterstained with hematoxylin.

Immunostaining of the pancreas with a commercially available goat polyclonal antibody against Fem1b demonstrates the same islet staining pattern, with an absence of specific staining in the Fem1b-KO homozygotes. Coimmunostaining with antibodies to insulin, a βcell marker, demonstrates that Fem1b is expressed in virtually all β cells (Figure 6).

Fig. 6. Immunofluorescence staining of insulin (green), Fem1b (red), and merged image demonstrating that Fem1b is expressed not only in insulin-positive β cells but also in insulin-negative non-β cells.

The coimmunostaining with antibodies to glucagon and somatostatin, markers for α cells and δ cells, respectively, demonstrates that the Fem1b protein is also expressed in these nonβ cells (Figure 7).

Fig. 7. Immunofluorescence staining with a combination of antibodies to glucagon and somatostatin (red) and Fem1b (green) and a merged image verifying expression of Fem1b within non- β cells.
