Donkey anti rabbit igg

Hearts were fixed in 4% PFA for 1 hour, washed in PBS, incubated in methanol/hydrogen peroxide (8:1) for 1 hour and then blocked in PBSST (5% Skim milk powder and 0.5%Triton X-100 dissolved in PBS) for five hours. The primary antibody used was Rabbit anti-mouse Connexin 40 (Alpha Diagnostic International, 1:200). Donkey anti-Rabbit IgG (Santa Cruz, 1:100) was used as a secondary antibody and visualization was performed by incubating with DAB substrate (Sigma Aldrich). All antibodies were diluted in PBSST and incubations were carried out at 4°C overnight. Following each overnight incubation, tissues were washed three time 1 hour each at 4°C with PBSST

Protein contents were assessed for the collected hippocampi supernatants, previously lysed in RIPA buffer containing protease inhibitor cocktail, via a BCA kit. Five μg of protein per sample was resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF) for western blotting. Membranes were blocked for 1h in 5% bovine serum albumin (BSA) in tris-buffered saline solution (TBST) followed by applying anti-amyloid precursor protein (1:1000 dilution, Y188, ab32136, rabbit mAb, mouse/rat/human cross-reactivity, Abcam, Cambridge, MA, USA), BACE (1:1000 dilution, D10E5, rabbit mAb, Cell Signaling Technology, Inc., Danvers, MA, USA), and Cox-2 (1:500 dilution, Cyclooxygenase 2, rabbit pAb, ab15191, Abcam, Cambridge, MA, USA), primary antibodies overnight at 4°C. Chemiluminescent visualization was used to detect antibody binding using donkey anti-rabbit IgG and goat-anti mouse IgM horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., CA, USA). Exposures were captured using an Aplegen Omega Lum G imaging system. Optical density values were normalized to the loading control α-tubulin (1:10000 dilution, TU-02: sc-8035, mouse mAb, Santa Cruz Biotechnology, Inc., CA, USA) optical density values.