Mouse anti p27

Five-micron thick sections from formalin-fixed, paraffin-embedded pancreases were prepared. Slides were rehydrated, blocked with CAS-block (Invitrogen) and incubated overnight at 4°C with primary antibodies. After washing in PBST slides were incubated for 1 h with fluorescent secondary antibodies, washed again, incubated briefly with Sytox Green Nucleic Acid Stain (Invitrogen) and mounted. Digital images of islets and acinar tissue were obtained using an Olympus BX51 microscope (200 X magnification), based on staining for insulin. The number of nuclei in a constant area (2500 μm²) was counted manually using Image-Pro Plus software (Media Cybernetics). The primary antibodies used were guinea pig anti-insulin (Dako), mouse anti-p27 (Santa Cruz), goat anti-PDX1 (a gift from Christopher VF Wright), rabbit anti-phospho rpS6(240/244) (Cell Signaling Technology, Beverly, MA, USA), and rabbit anti-Ki67 (Thermo Scientific). The secondary antibodies used were CY2-anti-guinea pig and CY3-anti rabbit (Jackson ImmunoResearch). Immunohistochemical staining of pancreatic sections for HA were carried out with mouse anti-HA (Cell Signaling Technology).

DMEM low glucose, fetal bovine serum, TNF-α, cell culture reagents, and CM-H₂DCFDA were purchased from Invitrogen (San Diego, CA). Primary antibodies, including mouse anti-cyclin D1, rabbit anti-CDK4, mouse anti-cyclin E, rabbit anti-CDK2, rabbit anti-p21, mouse anti-p27, rabbit anti-MMP2, and rabbit anti-MMP9, were purchased from Santa Cruz Biotechnology (CA, USA). Goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Enzo (Farmingdale, USA).