Ab53165

After 6 rounds of plaqued selection, 9 selected vaccinia VG9/(SST-14)₂-HSA were propagated in BSC-40 cells. Then, the infected cells and supernatant were harvested and lysed through 3 freeze-thaw cycles. The lysates of vaccinia were separated by 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 3% BSA and incubated with primary antibodies against HSA (ab83465, Abcam, London, UK), somatostatin (ab53165, Abcam) and β-actin (Santa Cruz, CA, USA). Finally, corresponding HRP-conjugated anti-IgG secondary antibodies were incubated and the blots were developed by an ECL luminol reagent (Santa Cruz).

The immunofluorescence procedures have been described by our group previously [18 (link)]. Blocked sections were incubated with a primary antibody mix at +4 °C, overnight, in a wet chamber. The following primary antibodies were used to stain the pancreas: anti-insulin (1:400, Abcam, Cambridge, MA, #ab7842), anti-glucagon (1:400, Abcam, #ab10988), anti-somatostatin (1:400, Abcam, #ab53165). Sections were rinsed in PBS 3 times, 10 min each, and incubated for 2 h at room temperature in a mix of fluorophore-conjugated secondary antibodies in wet chamber protected from light. Appropriate secondary antibodies were used that were conjugated with DyLight 488 (1:400, Jackson ImmunoResearch Laboratories Inc., West Grove, PA), Alexa 555 (1:400, Molecular Probes, Eugene, OR), or Alexa 647 (1:400, Molecular Probes). The same solution was used to dilute primary and secondary antibodies: 1% NDS, 1% BSA, 0.03% Triton X-100. After incubation with secondary antibody, slides were washed in PBS 3 times, 10 min each, and mounted with anti-fading agent Gel/Mount (Biomeda, Foster City, CA). Insulin, somatostatin, and glucagon were labelled by green, blue, and red fluorescent probes, respectively.
Stained slides were viewed using a Nikon C1Si microscope. Images were captured on a Nikon C1Si or C1 Plus confocal microscope, and were analyzed using Nikon software EZ-C1 3.90 Free viewer.