Anti doublecortin dcx

Cells were fixed with cold methanol for 5 min at room temperature and discarded. Subsequently, cell membrane permeabilization was performed by washing 3 times for 5 min with PBST (0.1% Tween 20 in PBS) and 10 min incubation with 0.1% Triton X-100 solution in PBST for 10 min at room temperature. After washing three more times with PBS, the blocking of nonspecific binding of antibodies was performed in a solution containing 1% bovine serum albumin (BSA) in PBST for 30 min. Cell cultures were then incubated with anti-NeuN antibodies (1: 200; Abcam, Cambridge, UK; Cat # ab6328) and anti-doublecortin (DCX) (1: 100; Sigma-Aldrich; Cat # ab52987) in 1% BSA in PBST at 4 °C overnight After washing thoroughly in PBS, cultures were embedded into mounting medium (3% glycerin in PBS) and examined under EVOS FL fluorescence microscopy.

After PET imaging, or 7 days after ar-turmerone treatment, rats were deeply anesthetized and decapitated. The brains were rapidly removed, frozen in isopentane, and stored at −80°C before further histologic and immunohistochemical processing. Ten-μm-thick adjacent serial coronal brain sections were cut at 500-μm intervals and stained with anti-BrdU to identify proliferating cells (mAb clone BU-33, dilution 1:200; Sigma-Aldrich), or with anti-doublecortin (DCX) to identify neuroblasts (rabbit polyclonal, dilution 1:1,000, Sigma-Aldrich). For antigen-retrieval before BrdU staining, sections were microwave-heated in 0.01 M citrate buffer, pH 6.0, for 5 minutes, followed by 2 N HCl at 37°C for 30 minutes. For visualization, the ABC Elite kit (Vector Laboratories with diaminobenzidine (Sigma-Aldrich) as the final reaction product was used.
To quantify the width of the SVZ and of the dentate gyrus of the hippocampus, it was measured on three consecutive BrdU-stained slices per animal, and an average was calculated per animal. To quantify the number of neuroblasts in the SVZ, their number was counted on three consecutive DCX-stained slices within a standardized field-of-view for each animal. For both schemes of quantification, mean values were calculated for each group of animals.