Monoclonal Anti-Proliferating Cell Nuclear Antigen antibody produced in mouse

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	-Mouse (1:1000)

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-Mouse (1:1000)

Publication protocol

On day 73, mice from all experimental conditions were deeply anesthetized for transcardial perfusion with 0.1 M phosphate-buffered saline pH 7.4 (PBS). Their brains were dissected out and cut through the midline. The left hemisphere was arbitrarily chosen for the histological study. It was post-fixed for 48 h in 4% paraformaldehyde in PBS and cut into coronal vibratome sections (50 µm) from −1.06 to −3.08 mm from bregma (Paxinos and Franklin, 2001), which includes the hippocampal area. For free-floating immunohistochemistry, sections first received a heat-induced epitope retrieval (EnVision Flex high pH solution; Dako, Glostrup, Denmark) followed by an endogenous peroxidase blocking (80% PBS, 10% methanol and 10% hydrogen peroxidase) for 30 min in the dark and incubated overnight with the corresponding primary antibody. A mouse anti-c-Fos antibody (1:500, Santa Cruz Biotechnology, sc-271243, lot 10413; which is also reactive for the c-Fos functional homologs Fos B, Fra-1 and Fra-2) was used for detection of basal neuronal activity; while the GABAergic interneuron populations were assessed by rabbit anti-PV (1:500; Swant, Marly, Switzerland, PV-28, lot 5.10), rabbit anti-NPY (1:500, Sigma, N9528, lot R40829) and rabbit anti-GABA (1:500, Sigma, A2052, lot 095K4830) antibodies. The AHN-related markers used were a mouse anti-proliferating cell nuclear antigen (PCNA; 1:1000, Sigma, P8825, lot 014M4836) to label cells undergoing proliferation in the dentate gyrus, and goat anti-doublecortin (DCX; 1:200, Santa Cruz, sc-8066, lot A1211) for immature neurons up to 3-4 weeks of age (Brown et al., 2003). All antibodies were diluted in PBS, 0.5% Triton X-100 and 2.5% donkey serum. On the second day, sections were incubated for 90 min in a biotin-conjugated secondary antibody (rabbit anti-mouse, rabbit anti-goat or swine anti-rabbit as appropriate; Dako; diluted 1:800) and for 1 h in peroxidase-conjugated extravidin (Sigma, 1:1000 in PBS) solution in the dark. The staining solution contained diaminobenzidine (DAB) as the chromogen (proportion: 0.1 ml of DAB previously diluted at 5% in distilled water, 10 µl hydrogen peroxidase and 10 ml PBS) and nickel chloride (NiCl2, Sigma) was added to intensify c-Fos labelling (0.004 g per 10 ml of staining solution). Each step was followed by PBS rinses. Negative controls in which the primary antibody was omitted resulted in absent staining.

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