Rabbit anti cnpase

Western blot analysis was performed as described previously [35 (link)], with slight modifications. Briefly, cells were lysed on ice with radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and sonicated to reduce sample viscosity. Protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA), equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Western blotting was performed using the following antibodies at 4 °C overnight: rabbit anti-β-III-tubulin (1:5000; Covance); rabbit anti-MAP-2 (1:2000; Millipore); rabbit anti-GFAP (1:5000; Abcam); rabbit anti-CNPase, rabbit anti-JNK, rabbit anti-p-JNK, rabbit anti-c-Jun, rabbit anti-p-c-Jun^ser63, and rabbit anti-p-c-Jun^ser73 (all 1:1000; all from Cell Signaling Technology); and mouse anti-β-actin (1:10,000; Sigma-Aldrich). Blots were incubated with horseradish peroxidase-labelled secondary anti-rabbit and anti-mouse antibodies, and immunoreactive bands were visualized on film by enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA) (all 1:10,000, Abcam). Western blots were quantified with ImageJ software from three independent experiments. The intensities of the bands were normalized to β-actin.

Brains were harvested and fixed in 4% PFA (pH7.4) for a day. The brains were then dehydrated for 24 hrs in 25% sucrose solution. All sections for KI67, DCX, c-Fos and CNPase staining were cut to a thickness of 30 μm on a sliding microtome. Sections were mounted on superfrost slides and dried overnight. Subsequently, slides were incubated in 0.01 mol/L citric buffer for 20 min at 90°C, 3% H₂O₂ for 10 min, rinsed in PBS, and incubated overnight at room temperature in rabbit anti-KI67 antibody (1:4000, Vector Lab), goat anti-DCX antibody (1:250, Santa Cruz), rabbit anti-c-Fos (1:1000, Santa Cruz), or rabbit anti-CNPase (1:1000, Abcam). The next day, a standard IgG ABC kit (Vector Lab) procedure was used and the slides reacted for 5–10 min with a Sigma DAB tablet. Sections were then counterstained with cresyl violet and cover-slipped with DPX.
For BrdU staining, following a 3% H₂O₂ incubation for 10 min, slides were subsequently incubated in 2M HCL for 30 min at 37°C. Rat anti-BrdU antibody (1:250, Accurate) was applied overnight. The next day the ABC kit procedure was followed and the slides were reacted with a Sigma DAB tablet. Hippocampus cells were counted bilaterally on every eighth section through the entire rostrocaudal extent of the granule cell layer.