Rabbit anti gapdh antibody

Metformin (1,1-dimethyl biguanide hydrochloride) was purchased from Sigma (#D150959, Sigma-Aldrich, St. Louis, MO, USA) and dissolved in a serum-free medium to a stock solution of 1 M. Other chemicals and reagents were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and Merck (Merck KGaA, Darmstadt, Germany).
Antibodies were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and CST (Cell Signaling Technology, Beverly, MA, USA). The antibodies used for Western blots were anti-rabbit IgG-peroxidase (#A0545, Sigma), rabbit anti-GAPDH antibody (#G9545, Sigma), rabbit anti-β-actin antibody (#A2066, Sigma), anti-PARP antibody (#9542, CST), anti-caspase-3 antibody (#9662, CST) and anti-cleaved caspase-3 antibody (#9664, CST).

For Western blot analysis, collected hippocampi were placed in RNAlater (Life Technologies) and homogenized in an SDS sample buffer. Protein extracts were separated by SDS–polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (Hybond P; GE Healthcare). The membrane was blocked with a blocking agent (GE Healthcare) and then incubated at 4°C overnight with a primary antibody for rabbit anti-HSP110 (1:5000, StressMarq Biosciences Inc.), rabbit anti-HSP25 (1:5000, Cosmo Bio Co. Ltd.), mouse anti-HSP60 (1:1000, Cosmo), mouse anti-HSP72 (1:1000, Cosmo), mouse anti–phospho-CREB (Ser¹³³) (1:5000, Cell Signaling Technology), rabbit anti-CREB (Ser¹³³) (1:5000, Cell Signaling), and rabbit anti-BDNF (1:5000, Abcam). After washing with tris-buffered saline containing 0.1% (v/v) Tween 20, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (1:20,000) for 1 hour at room temperature. The antibody-reactive bands were visualized using the chemiluminescent substrate kit (GE Healthcare). Bands were analyzed by densitometry using FluorChem 8800 (Alpha Innotech), and the content of GAPDH, which was detected using a rabbit anti-GAPDH antibody (1:20,000; Sigma), was used as a control to ensure that the same amount of protein was loaded in each lane.