Rabbit anti somatostatin

Images of the mice and their organs were obtained using a digital fluorescent microscope (VB-6000; Keyence Japan, Osaka, Japan, or Leica AF6500; Leica Microsystems, Tokyo, Japan). For histological analyses, tissues were fixed in 10% formalin solution and embedded in paraffin for hematoxylin and eosin (H&E) staining. Alternatively, tissues were fixed by perfusion with 4% paraformaldehyde. Frozen sections were the cut using a Microm HM500 OM Microtome Cryostat (Carl Zeiss Japan, Tokyo, Japan).
The following primary antibodies were used for immunohistochemical analysis: guinea pig anti-insulin, rabbit anti-glucagon, rabbit anti-somatostatin, goat anti-pancreatic polypeptide, rabbit anti-chromograninA (cgA), rabbit anti-Ki67, and rabbit anti-VEGF (Abcam, Tokyo, Japan). Alexa568- or Cy3- labeled species-specific anti-IgG antibodies (Life Technologies Japan, Tokyo, Japan) were used as secondary antibodies. Images were obtained using a LSM710 laser scanning microscope (Carl Zeiss Japan), or a HS BZ-9000 fluorescent microscope system (Keyence). The Ki-67 index was calculated by dividing the total number of nuclei by the number of Ki-67-positive nuclei. The number of nuclei was counted by observing 8–10 fields with a 40× lens.

FFPE pancreas sections of 4 µm thickness were dewaxed and rehydrated according to standard protocols. The sections were immersed in a sodium citrate buffer (10 mM sodium citrate, pH 6.0) and subjected to heat-induced epitope retrieval. After rehydration in PBS, the sections were treated with a serum-free protein block (Dako, North Sydney, NSW, Australia) for 20 min, followed by primary antibody incubations at 4 °C overnight using guinea pig anti-insulin (1:200, Abcam, Melbourne, VIC, Australia), mouse anti-glucagon (1:200, Abcam, Melbourne, VIC, Australia), and rabbit anti-somatostatin (1:250, Abcam, Melbourne, VIC, Australia), all diluted in antibody diluent (Dako, North Sydney, NSW, Australia). Visualisation was performed using Alexa Fluor dyes (Life Technologies, Scoresby, VIC, Australia) and was imaged using a confocal Nikon A1+ point scanning confocal microscope.