Ab184955

Samples of collected tissues and cultured cells were prepared into a homogenate and centrifuged to collect the protein supernatant. Then, after quantifying protein concentrations by using a BCA assay kit (Pierce Biotechnology, Rockford, IL), an appropriate amount of sample protein was resolved by 10% denaturing SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA), which were then blocked at room temperature for 1 h by using an Odyssey blocking reagent (Li-Cor, Lincoln, NE), incubated overnight in a 4 o C fridge with anti-SIN3A primary antibodies (ab129087, 1:1000 dilution, Abcam, Cambridge, MA), anti-Arc primary antibodies (ab18950, 1:1000 dilution, Abcam, Cambridge, MA), anti-Egr1 primary antibodies (ab133695, 1:1000 dilution, Abcam, Cambridge, MA), anti-Homer1 primary antibodies (ab184955, 1:1000 dilution, Abcam, Cambridge, MA) and anti-Narp primary antibodies (ab191563, 1:1000 dilution, Abcam, Cambridge, MA) and further incubated with HRP-conjugated secondary antibodies (ab6721, 1:2000 dilution, Abcam, Cambridge, MA). The relative protein expression was analyzed using an Odyssey imaging system (Li-Cor, Lincoln, NE).

Samples of collected tissues and cultured cells were prepared into a homogenate and centrifuged to collect the protein supernatant. Then, after quantifying protein concentrations by using a BCA assay kit (Pierce Biotechnology, Rockford, IL), an appropriate amount of sample protein was resolved by 10% denaturing SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA), which were then blocked at room temperature for 1 h by using an Odyssey blocking reagent (Li-Cor, Lincoln, NE), incubated overnight in a 4 o C fridge with anti-SIN3A primary antibodies (ab129087, 1:1000 dilution, Abcam, Cambridge, MA), anti-Arc primary antibodies (ab18950, 1:1000 dilution, Abcam, Cambridge, MA), anti-Egr1 primary antibodies (ab133695, 1:1000 dilution, Abcam, Cambridge, MA), anti-Homer1 primary antibodies (ab184955, 1:1000 dilution, Abcam, Cambridge, MA) and anti-Narp primary antibodies (ab191563, 1:1000 dilution, Abcam, Cambridge, MA) and further incubation with HRP-conjugated secondary antibodies (ab6721, 1:2000 dilution, Abcam, Cambridge, MA). The relative protein expression was analyzed by using an Odyssey imaging system (Li-Cor, Lincoln, NE).

Samples of collected tissues and cultured cells were prepared into a homogenate and centrifuged to collect the protein supernatant. Then, after quantifying protein concentrations by using a BCA assay kit (Pierce Biotechnology, Rockford, IL), an appropriate amount of sample protein was resolved by 10% denaturing SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA), which were then blocked at room temperature for 1 h by using an Odyssey blocking reagent (Li-Cor, Lincoln, NE), incubated overnight in a 4 o C fridge with anti-SIN3A primary antibodies (ab129087, 1:1000 dilution, Abcam, Cambridge, MA), anti-Arc primary antibodies (ab18950, 1:1000 dilution, Abcam, Cambridge, MA), anti-Egr1 primary antibodies (ab133695, 1:1000 dilution, Abcam, Cambridge, MA), anti-Homer1 primary antibodies (ab184955, 1:1000 dilution, Abcam, Cambridge, MA) and anti-Narp primary antibodies (ab191563, 1:1000 dilution, Abcam, Cambridge, MA) and further incubated with HRP-conjugated secondary antibodies (ab6721, 1:2000 dilution, Abcam, Cambridge, MA). The relative protein expression was analyzed using an Odyssey imaging system (Li-Cor, Lincoln, NE).

After the selection of DG-and BLA-containing tissue sections, washings with PBS and antigen retrieval technique proceedings were carried out as described previously. Unspecific bindings were blocked with BSA solution (3 % in 0.3 % of Triton-X-100 in PBS for 90 min, RT). Sections were incubated for 48 h at 4 ºC with gentle agitation with guinea pig polyclonal anti Arc (156 004;
1:1500; Synaptic Systems ® , Goettingen, Germany), mouse monoclonal anti GluN1 antibody (32-0500; 1:100; ThermoFischer, Massachusetts, USA) and rabbit monoclonal anti Homer1 (ab184955; 1:150; Abcam). Later, brain tissue samples were incubated with the following secondary antibodies: anti-mouse conjugated with Alexa Fluor 555 (A-31570, 1:1000, Invitrogen), anti-guinea pig conjugated with Alexa 488 (A-11073, 1:1000, Invitrogen) and anti-rabbit conjugated with Alexa 647 (A-31573; 1:1000, Invitrogen) for 4 h, as described in the previous section. Sections were also incubated with DAPI (1:25000) for 1 min and mounted with ProLong® Gold (Invitrogen) in gelatinized slides.