Ab15317

Pancreata were fixed overnight in 4% formaldehyde and embedded in paraffin. Sections were rehydrated and heated for 20 min in Target Retrieval Solution (pH 6.1, Dako). After blocking with 20% normal goat serum (Dako) in PBS, slides were incubated with 1/1,000 anti-hGH monoclonal antibody (ab15317, Abcam) in Antibody Diluent (Dako). For double immunofluorescent labeling, 1/50,000 rabbit anti-serotonin (Immunostar, #20080) or 1/2,000 polyclonal anti-GLUT2 (07-1402, Millipore) was combined with 1/10,000 diluted guinea pig anti-insulin antibody (a gift of Dr. Van Schravendijk, VUB, Brussels) and detected with anti-rabbit Cy3 and anti-guinea pig FITC, respectively (both from Jackson ImmunoResearch Laboratories). For MIP-GFP and RIP-Cre mice, frozen sections were stained with 1/10,000 rabbit anti-serotonin or 1/200 mouse anti-hGH (blocking with Mouse on Mouse [M.O.M.] Basic Kit from Vector Laboratories) and costained with 1/2,000 guinea pig anti-insulin in sections from RIP-Cre mice. For MIP-GFP, direct fluorescence for GFP was used to detect β cells.

Islets were isolated as described above and homogenized in lysis buffer (Cell Signaling Technology) by sonication. Protein extracts were separated by SDS-PAGE (10% [v/v] Bis/Tris gel; Life Technologies), blotted on a nitrocellulose membrane, blocked in 4% (w/v) milk, and incubated with primary antibody (anti-hGH, ab15317, 1/1,000; anti-GAPDH, 1/15,000; clone 6C5, both from Abcam). The blot was subsequently incubated with peroxidase-conjugated secondary antibody (Dako), and proteins were detected using the Western Lightning ECL System (PerkinElmer). For hGH staining on islets from MIP-GFP and RIP-Cre mice, a similar protocol was used with adjustments: 10 μg of islet protein sample in 1× Laemmli sample buffer was resolved on 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore).