Anti pa

Hepatic and pancreatic tissue lysates from control and STZ-induced male and female rats containing equal amounts of protein were separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked for 1h with 5% skim milk at room temperature, and incubated with the indicated primary antibodies for 2 h at 1:1000 dilution (anti-β-actin, anti-SPARC, anti-C/EBPβ, anti-ATP5B, anti-CD95, anti-MCP1, anti-iNOS, anti-Cyt C, anti-SOD2, anti-INS, anti-CA3, anti-RARhoGAP, anti-CPS1, anti-BHMT, anti-PA, anti-APC2 [Santa Cruz Biotechnology, Santa Cruz, CA, USA], anti-TNFα, anti-PARP-1, anti-NFκB, anti-HSP90 [Cell Signaling Technology, Beverly, MA, USA], and anti-CRP [AbFrontier, Seoul, Korea]). After washing with Tris-buffered saline containing Tween 20, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Then, immune complexes were detected using the ECL method, and immunoreactive bands were quantified by densitometric analysis using ImageMaster 2D software version 4.95 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Relative intensity (%) values of proteins were normalized to β-actin levels.

For the histological study, pancreatic and hepatic tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin wax. Paraffin-embedded tissue sections (4 μm each) were deparaffinized, rehydrated, and subjected to hematoxylin and eosin staining. Immunohistochemistry was performed on formalin fixed, paraffin-embedded hepatic and pancreatic tissues. The sections were then incubated with primary antibodies (anti-SPARC, anti-CA3, anti-CPS1, anti-RARhoGAP, anti-BHMT, anti-PA, anti-APC2 [dilution 1:100, Santa Cruz Biotechnology], and anti-insulin [dilution 1:400, Cell Signaling Technology]) overnight at 4°C, followed by incubation with appropriate HRP-conjugated secondary antibodies at room temperature for 2 h. The immunoreactivity was visualized with diaminobenzidine (DAB) staining (Vector Laboratories, Burlingame, CA, USA), and then counterstained with Mayer's hematoxylin (Vector Laboratories). The histopathological findings were observed using light microscopy (X400; Olympus IX51, Tokyo, Japan).