Horseradish peroxidase hrp conjugated anti rabbit secondary antibody

Cells were scraped and resuspended in 40 volumes of PBS (10 mM phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.6) with protease inhibitor cocktail (Roche, Milan, Italy) and forced 40 times through a 27-gauge needle. They were then centrifuged for 15 min at 6000 rpm at room temperature. The supernatant was collected, and the pellet was washed again with the same volume of PBS and centrifuged as above twice. The second and third supernatants were discarded and the pellet resuspended in the same volume of PBS. The protein extraction from the 30 mg biopsy was performed as above in 40 volumes of PBS. Pellets and the first supernatant were run on a 12% polyacrilamide gel, then blotted on nitrocellulose membrane, blocked for 60 min with 1.5% bovine serum albumin, and incubated overnight with primary antibodies against: PKA catalytic subunit, PKA RIIA subunit, or PKA RIB subunit (made in rabbit, Santa Cruz Biotechnology, DBA Segrate, Italy, 1:5000 in 1.5% bovine serum albumin), CD133 (made in mouse, Miltenyi, Calderara di Reno, Italy 1:10,000 in 1.5% bovine serum albumin) or nestin (made in goat, Santa Cruz Biotechnology, 1:10,000 in 1.5% bovine serum albumin), incubated for 2 h with horseradish peroxidase (HRP)-conjugated antirabbit secondary antibody (Sigma, Milan, Italy, 1:10,000) and revealed through chemiluminescence (Advanced ECL, Amersham, Milan, Italy).

Human hippocampal protein was extracted in 0.01 M phosphate buffer containing a protease inhibitor cocktail (Sigma) using mechanical homogenization and 3 freeze–thaw cycles. Homogenates were centrifuged for 10 min at 12,000 rpm at 4 °C. Total protein levels in each sample were quantified by the BCA assay (Pierce) and 50 μg total protein per lane was loaded onto a NuPAGE Novex Tris–acetate 3–8 % gradient mini-gel (Life Technologies). After separation, proteins were transferred onto a Hybond ECL nitrocellulose membrane (GE Healthcare) using a Mini Trans-Blot Cell (Bio-Rad) for 4 h and loading was checked with Ponceau S (Sigma). Membranes were probed with the primary antibody that was to be used for anti-RAI1 immunohistochemistry, rabbit anti-RAI1 (1:1000; Abcam). Labelled proteins were detected using a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Sigma; 1:5000) and enhanced chemiluminescence (ECL; Millipore), incubated for 5 min followed by exposure to X-ray film (Thermo Fisher Scientific).