Goat anti dcx antibody

For labeling of proliferating cells, 50 mg/kg body weight of 5-bromo-20-desoxyuridine (BrdU; Sigma Aldrich) was injected i.p. daily for five following days starting at the peak of disease (14 d.p.i.). EAE animals and healthy controls were euthanized and perfused 2 weeks after the first BrdU-injection. Mouse hippocampi were collected and cryopreserved (see histology part) and afterwards processed as previously described [16 (link)]. Briefly, sections were incubated in 2 N HCl for 30 min at 37 °C and rinsed with 100 mM tetraborate buffer. Sections were incubated with primary rat anti-BrdU antibody (1:400; Upstate) and goat anti-Dcx antibody (1:500; Santa Cruz Biotechnology) overnight at 4 °C, followed by incubation with appropriate Cy2/Cy3-conjugated secondary antibodies (Millipore) for 1 h at RT.

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.

To visualize cell differentiation, we performed immunohistochemistry for DCX in the dentate of the hippocampus. The sections were incubated in PBS for 10 min, washed three times for 3 min in PBS, and incubated in 1% H₂O₂ for 15–30 min. The sections were selected from each brain and incubated overnight with goat anti-DCX antibody (1:500; Santa Cruz Biotech, sc-8066). The following day, the sections were incubated with biotinylated rabbit secondary antibody (1:250; Vector Laboratories) for 90 min. The secondary antibody was amplified with the Vector Elite ABC kit^® (1:100; Vector Laboratories). Antibody-biotin-avidin-peroxidase complexes were visualized using a DAB substrate kit (vector Laboratories). The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount ^®.

To obtain accurate data for immunohistochemistry, the free-floating sections from all animals were processed carefully under the same conditions. For each animal, tissue sections were selected from between 1.46 mm and 2.46 mm posterior to the bregma by referring to the mouse atlas by Franklin and Paxinos [24 ]. Ten sections, 90 μm apart, were sequentially treated with 0.3% hydrogen peroxide (H₂O₂) in PBS for 30 min and 10% normal goat or rabbit serum in 0.05 M PBS for 30 min. They were then incubated with a rabbit anti-Ki67 antibody (1 : 1000; Abcam, Cambridge, UK) or goat anti-DCX antibody (1 : 50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 25°C and subsequently treated with either a biotinylated goat anti-rabbit IgG or a rabbit anti-goat IgG and a streptavidin-peroxidase complex (1 : 200, Vector Labs, Burlingame, CA, USA). Sections were visualized by reaction with 3,3′-diaminobenzidine tetrachloride (Sigma) in 0.1 M Tris-HCl buffer (pH 7.2) and dehydrated and mounted in Canada balsam (Kanto Chemical, Tokyo, Japan) onto gelatin-coated slides.

BrdU immunofluorescent labeling was obtained with a 1:500 dilution of the rat anti-BrdU antibody (Accurate Chemical and Scientific Corporation OBT0030, Westbury, NY, USA) and doublecortin (DCX) immunohistochemical labeling was obtained with a 1:800 dilution of the goat anti-DCX antibody (SantaCruz Biotechnology, Dallas, TX, USA). Immunolabeling for both antibodies followed previous descriptions (Naninck et al., 2015 (link); Hoeijmakers et al., 2018 (link)).