Immunohistochemistry was performed, as previously described (9 (link), 24 (link), 110 (link)). In brief, pancreas samples were fixed in 4% paraformaldehyde and paraffin embedded. Sections were deparaffinized in a xylene ethanol series and placed in Tris-EDTA (TE) buffer for antigen retrieval (10 mM Tris, 1 mM EDTA, 0.05% Tween, pH = 9.0) and then blocked in 5% BSA. Sections were immunostained for insulin using a monoclonal anti-mouse antibody (Santa Cruz Biotechnology, SC-377071; 1:100) and for glucagon using a monoclonal anti-rabbit antibody (Santa Cruz Biotechnology, SC-7779R; 1:200). Separate sections were immunostained for GLP-1 using a monoclonal anti-mouse antibody (Abcam, ab26278; 1:100) and glucagon using a monoclonal anti-rabbit antibody (Santa Cruz Biotechnology, SC-7779R; 1:200). Separate sections were immunostained for PC1/3 using a monoclonal anti-mouse antibody (Abcam, ab3532; 1:200) and glucagon using a monoclonal anti-rabbit antibody (Santa Cruz Biotechnology, SC-514592; 1:200). Detection of primary antibodies was performed with Alexa Fluor 488 anti-rabbit (A11034), Alexa Flour 488 anti-mouse (A11001), Alexa Flour 633 anti-mouse (A21052), and Alexa Fluor 633 anti-rabbit (A21070) secondary antibodies (1:200) (Invitrogen). Nuclei were detected with DAPI (Invitrogen, P36962).
Saikia M., Holter M.M., Donahue L.R., Lee I.S., Zheng Q.C., Wise J.L., Todero J.E., Phuong D.J., Garibay D., Coch R., Sloop K.W., Garcia-Ocana A., Danko C.G, & Cummings B.P. (2021). GLP-1 receptor signaling increases PCSK1 and β cell features in human α cells. JCI Insight, 6(3), e141851.
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