Rat anti brdu

Asynchronously growing DT40 cells were sequentially labelled for 15 min with 25 μM IdU and for 15 min with 25 μM CIdU. UV treated cells were irradiated at 20 J/m² just before the CldU treatment. At the end of the labelling period (30 min), cells were placed in ice cold 1× PBS (1 volume of cells for 2 volumes of 1× PBS) and centrifuged at 250 g for 5 min at 4°C, washed in ice-cold PBS, and resuspended in PBS to a final concentration of 1 × 10⁶ cells/ml. Three microliters of the cell suspension was spotted onto clean glass Superfrost slides and lysed with 7 μl of 0.5% SDS in 200 mM Tris–HCl (pH 5.5) and 50 mM EDTA (5 min, at room temperature). Slides were tilted at 15° to horizontal, allowing the DNA to run slowly down the slide. Slides were then air dried and fixed in 3:1 methanol/acetic acid, and stored at 4°C before immunolabelling. IdU, CldU, DNA revelations and analysis were performed as described (26 (link)), with minor modifications: the DNA was denatured for 30 min in 2.5 N HCl, and CldU was detected using rat anti BrdU (ABD Serotec, Raleigh, NC, USA) at 1/750. A stretching factor of 2.6 for conversion from μm to kb was applied, as previously described for the method in (27 (link)). Slides were mounted in 10% 1× PBS and 90% glycerol, kept at −20°C and imaged using a Nikon C1-si confocal microscope.

A set of new sections and selected sections stained with NADPH-d histochemistry described above were simultaneously processed for BrdU immunolabeling with the DAB-peroxidase method. Sections were pre-treated in 1 × SSC and 50% formamide for 1 h at 65°C, in 2N HCl for 30 min at 37°C, and in PBS containing 1% H₂O₂, 5% normal rabbit serum and 0.3% Triton X-100 for 45 min. Sections were next incubated overnight at 4°C with rat anti-BrdU (AbD Serotec, Raleigh, NC, USA, MCA2060, 1:2000) diluted in PBS containing 5% rabbit serum, reacted with biotinylated rabbit anti-rat IgG at 1:400 for 2 h, and subsequently with the ABC reagents (1:400) (Vector Laboratories, Burlingame, CA, USA) for an additional hour. Immunoreactivity was visualized in 0.003% H₂O₂ and 0.05% diaminobenzidine.

In order to identify the neuronal phenotype of the new cells that reach the granular cell layer (GrL), double staining for BrdU, and the neuronal protein NeuN was used. Following the protocol previously described (Corona et al., 2011a (link); Portillo et al., 2012 (link); Arzate et al., 2013 (link)), OB slices were incubated for 48 h at 4°C simultaneously with both primary antibodies rat anti-BrdU (1:800, AbD Serotec) and mouse anti-NeuN (1:250, MILLIPORE) and then, after washing, with secondary antibodies anti-rat IgG Alexa Fluor 488 (1:1250, Invitrogen) and anti-mouse IgG Alexa Fluor 568 (1:1250, Invitrogen), respectively. After rinsing, sections were mounted on slides and coverslipped using aqua poly/mount, a non-fluorescing aqueous mounting medium (Polysciences, Inc).