Biotinylated goat anti rat secondary antibody

BrdU-labelled cells were detected by immunohistochemistry as previously described^{8 (link), 33 (link)}. Briefly, after denaturing the DNA (incubation of 1 hour at 37 °C in HCl 2 N), sections were subjected to a standard protocol of immunelabeling revealed with 3,3′-diaminobenzidine (see supplementary material). The antibodies used were an anti-BrdU monoclonal rat primary antibody (1:5,000; Abcam, Cambridge, UK) and a biotinylated goat anti-rat secondary antibody (1:300; Jackson, West Grove, PA, USA).
For determining the neuronal fate of BrdU positive cells a double immunofluorescent technique was carried out (see supplementary material). In each reaction the anti-BrdU monoclonal rat antibody (1:5,000; Abcam) was combined with either rabbit anti-calbindin D-28k (1:2,000; Swant, Bellinzona Switzerland), rabbit anti-calretinin (1:2,000; Swant), rabbit anti-GAD67 (1:1,000; Merck-Millipore, Darmstadt, Germany), rabbit anti-parvalbumin (1:2,000; Swant), mouse anti-reelin (1:1,000; Millipore, Temecula CA, USA), rabbit anti-somatostatin (1:3,000; Swant) or mouse anti-tyrosine hydroxylase (1:10,000; Jacques Boy, Reims, France) antibodies. The correspondent secondary fluorescent antibodies were Cy2-conjugated goat anti-rat antibody (for BrdU-labelled cells; 1:500; Jackson), and other Cy3-conjugated goat antibodies (for the other antigens studied; 1:500; Jackson).

One series of ten brain sections of animals injected with BrdU at P19, P21 and P30 were subjected to double/triple immunostaining. After denaturation and blocking as described above, the sections were incubated overnight in the mouse anti-CC1 (APC) (1:100, Abcam, Cambridge, UK) and rat anti-BrdU (1:500, Abcam, Cambridge, UK) primary antibodies. Appropriate secondary antibodies, namely goat anti-rabbit 568 (1:500, Abcam, Cambridge, UK) and biotinylated goat anti-rat secondary antibody (1:200, Jackson ImmunoResearch, Cambridgeshire, UK), followed by Alexa Fluor 488-conjugated streptavidin (1:500, AlexaFluor Invitrogene, Waltham, MA, USA), were subsequently used. Next, the brain sections, after rinsing with PBS, were counterstained with DAPI (1:5000, Sigma-Aldrich, Darmstadt, Germany). Finally, the brain sections were mounted on glass slides and coverslipped with 60% glycerol in PBS.