Western Blot

A separate group of animals (n = 18) were used for western blot experiments (trace-conditioned: n = 6; delay-conditioned: n = 6 and un-conditioned control: = 6). The trace- and delay-conditioned animals were sacrificed 1 h after the training, with xylazine (10 mg/kg) and ketamine (85 mg/kg) overdose, and the brain was extracted. The un-conditioned controls animals were also sacrificed similarly at a time-matched hour. The hippocampus was removed, and one side was kept intact, while the other side was further dissected into the DH and VH. The selection for keeping one side of the hippocampus intact and dissection of the other side was done randomly. The hippocampal tissue was dipped in the lysis buffer [RIPA (3 ml/gm), in which phenylmethyl sulfonyl fluoride (PMSF; 1 mM) and Protease Inhibitor Cocktail (PIC; 1:100)] were also added. The tissue was incubated for 10 min, and thereafter it was homogenized on ice. Tissue lysate was then centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected and stored at −80°C for further analysis.

Electrophoresis was performed as per the standard protocol. In brief, the total protein content in each sample was quantified using the Bradford assay. Samples for western blot were prepared. An equal amount of protein (50 μg per well) was loaded and resolved in 10% SDS-PAGE (BioRad western unit). Proteins were electro-blotted onto the PVDF membrane at 10V for 30 min (TransBlot semidry, BioRad). The membrane was blocked with 5% BSA in Tris-buffered saline (TBS) for 2 h at room temperature. After blocking, the membrane was washed with TBST (TBS with 0.5% Tween-20) and then incubated with primary antibody solution (in TBST) overnight at 4°C. After primary incubation, the membrane was washed with TBST (four times) and then incubated in secondary antibody for 3 h at room temperature. After washing, protein bands were visualized using the chemiluminescence method [ECL (Abbkines)] in ChemiDoc (Bio-Rad ChemiDoc TM XRS⁺ systems) using “Quantity One” software (Bio-Rad, USA).

Primary antibodies used for western blot analysis were, anti-CREB (1:1,000, Abcam), anti-pCREB (1:1,000, Abcam), anti-ErK1&2(1:1,000, Abcam), anti-pErk1&2 (1:1,000, Abcam), anti-Arc (1:2,000, Abcam), and anti β-actin (1:2,500, Sigma–Aldrich). Secondary antibodies used for the western blot analysis were as follows; goat anti-rabbit polyclonal HRP-tagged (1:10,000, Santa Cruz Biotechnology) and goat anti-mouse polyclonal HRP-tagged (1:10,000, Santa Cruz Biotechnology). The pre-stained protein ladder (Genedirex) was used for the identification of desired protein bands.