Anti rabbit igg hrp secondary antibody

Cells were lysed with NP-40 lysis buffer and protein concentration was determined by Bradford assay. Samples were subjected to gel electrophoresis and transferred to PVDF membrane by dry transfer (iBlot; Invitrogen). Membranes were blocked for 1h with 5% BSA. Primary antibodies for p-STAT5 (Y694), GAPDH (14C10), BCL-xL, BCL-2 (D55G8), MCL-1 (D5V5L) and cleaved caspase 3 (D175) (Cell Signaling Technology) and STAT5 (sc-835, Santa Cruz Biotechnology) were used. Primary antibodies were diluted 1:1000 in 1% BSA and incubated overnight at 4°C. Anti-rabbit IgG HRP secondary antibody (Cell Signaling Technology) diluted 1:2000 was incubated for 1h at room temperature. Blots were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare) and imaged/analyzed using Image Lab software (BioRad).

Mice were anaesthetized with isoflurane and perfused with 4% paraformaldehyde prepared in PBS (0.1 M, pH=7.5). Brain and pancreas tissues were isolated, fixed in 10% neutral formalin, dehydrated in a graded series of ethanol, embedded in paraffin, and cut into 5-μm slices using a slicing machine (Leica, Germany). For immunofluorescence, slides after deparaffinization and rehydration were placed in citric acid buffer (10 mM, pH=6.5) within a decloaking chamber at 120 °C for 5 min and cooled at room temperature for 1 h. Then, slides were washed two times in PBS for 5 min, treated with 3% H₂O₂ for 30 min and blocked with 5% BSA for 30 min at 37 °C. Sections were incubated with primary antibody SYP (1:200, cell signal technology) at 4 °C overnight. After triple washing with PBS (5 min/time), sections were incubated with anti-rabbit IgG-HRP secondary antibody (1:500, cell signal technology) at 37 °C for 1 h. Sections were washed in PBS (5 min/time) and added with DAPI (200 ng/ml) for 2 min at room temperature. Pancreas sections were incubated with primary antibody anti-insulin (Abcam, ab181547) and anti-glucagon (Abcam, ab10988) overnight at 4 °C and then incubated with secondary antibody (1:500, anti-rabbit IgG-HRP) at 37 °C for 1 h. Finally, the images were captured by using a Nikon ECLIPSE Ti microscope and processed by the ImageJ software.

Proteins were extracted from hippocampal tissue using RIPA buffer containing proteinase (Complete, Roche) and phosphatase inhibitors (1 mM β-glycerophosphate, 10 mM NaF, and 0.1 mM Na₃VO₄). Total protein was determined using a BCA protein assay kit, run on polyacrylamide gel, and transferred on to a PVDF membrane (Millipore, Billeria). The membrane was blocked with 10% milk in PBS for 1 h at room temperature and incubated overnight at 4 °C with synaptophysin (SYP) (1:1000, cell signal technology) and anti-α-tublin (1:1000, Abcam) antibodies. After washing five times with TBST, the membrane was incubated with anti-rabbit IgG-HRP secondary antibody (1:10000, cell signal technology) at room temperature for 1 h. Proteins were detected with a chemiluminescence (ECL) kit (Millipore, Billeria). Then, the relative density of protein bands was scanned using an LAS 4000 Fujifilm imaging system (Fujifilm, Tokyo, Japan) and analyzed by Quantity-One software (Bio-Rad Hercules, CA, USA).