Rat monoclonal anti brdu

To detect GFP, phospho-Paxillin, Vinculin or Myogenin, larvae were anesthesized in 0.02% MS-222, fixed in 4% paraformaldehyde (PFA, Sigma) overnight at 4°C and subsequently stored in 100% methanol (Fisher) at −20°C. Samples were washed in 0.1% Tween20 in phosphate-buffered saline (PBT, Sigma) and permeabilized. Samples were blocked in 5% goat serum (Life Technologies) in PBT for at least 2 h, then incubated with primary antibody diluted in 5% goat serum/PBT over night at 4°C. Samples were washed for at least 4 h in 0.1% PBT the following day, then incubated with secondary antibodies diluted in 5% goat serum/PBT for 2 h at room temperature. After several washes in PBT, samples were taken through glycerol series and mounted in VectaShield with DAPI (Vector Laboratories).
To label dividing cells with BrdU, larvae were exposed to 10 mM BrdU diluted into E3 medium and processed as previously described [16 (link)].
Antibodies used were chicken polyclonal anti-GFP (1 : 500; Millipore), rabbit polyclonal anti-GFP (1 : 500; Life Technologies), rabbit polyclonal anti-phospho-Paxillin (Tyr118) (1 : 200; Thermo Scientific), mouse anti-Vinculin (1 : 200; Sigma), rabbit polyclonal anti-Myogenin (1 : 50; Santa Cruz Biotechnology), rat monoclonal anti-BrdU (1 : 250; Abcam). Secondary antibodies used were Alexa conjugated antibodies diluted 1 : 500 in goat serum and PBT.

We used the following primary antibodies: mouse monoclonal anti-PV (1:2000, Sigma-Aldrich), CB (1:2000, Sigma-Aldrich), calretinin (CR; 1:10000, Millipore, Billerica, MA, USA), and glutamate decarboxylase 67 (GAD67) (1:10,000, Millipore); rat monoclonal anti-BrdU (1:100; Abcam, Cambridge, MA, USA); rabbit polyclonal anti-gamma-aminobutyric acid (GABA; 1:1000, Sigma-Aldrich) and anti-Ki67 (1: 10, Ylem, Rome, Italy), and neuropeptide Y (NPY, 1: 2000, Sigma-Aldrich); and goat polyclonal anti-DCX (1:200, Santa Cruz Biotechnology, Dallas, TX, USA). We also used the following secondary antibodies: Alexa Fluor 488 and 594 goat anti-mouse IgG (both 1:200, Life Technologies, Carlsbad, CA, USA), Cy3 goat anti-mouse IgM (1:200, Millipore), and Alexa Fluor 594 goat anti-rabbit IgG and anti-rat IgG (both 1:200, Life Technologies). Biotinylated Wisteria floribunda agglutinin (1:200, Sigma-Aldrich), followed by Alexa Fluor 488 conjugated to streptavidin (10 μg/ml), was used to label PNNs using the method described above [12 (link)].

Brain processing and immunohistochemical detection of the proliferation marker BrdU, the astrocyte and neural stem cell marker GFAP, the neural progenitor cell marker nestin, the early neuronal differentiation marker doublecortin (DCX), and the mature neuron maker NeuN were performed as previously described^{49 (link)}.
Primary antibodies used were mouse monoclonal anti-BrdU (1:100) from Dako (Hamburg, Germany) or rat monoclonal anti-BrdU (1:100) from Abcam (Cambridge, UK), mouse polyclonal anti-GFAP both from Cell Signaling (Beverly, MA, USA), goat polyclonal anti-DCX (1:500), goat polyclonal anti-nestin (1:500), and mouse monoclonal anti-NeuN (1:100) all of them from Abcam (Cambridge, UK); goat anti-ChAT, polyclonal, 1:100, Merk Millipore (Billerica, MA, USA) mouse anti-parvalbumin, monoclonal, 1:100, Merk Millipore (Billerica, Ma, USA). Secondary antibodies used were Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-mouse, Alexa Fluor 405 goat anti-mouse, Alexa Fluor 594 donkey anti-rat, Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 594 donkey anti-rabbit and Alexa Fluor 594 donkey anti-goat (all at 1:1000, from Life Tech).

Mice were anesthetized with chloral hydrate and perfused transcardially with ice-cold saline followed by perfusion with 4% paraformaldehyde 24 h after reperfusion. The brains were removed and post-fixed overnight for 24 h in paraformaldehyde; they were then coronally sectioned (30 μm) using a vibrating microtome (Leica, Wetzlar, Germany). The sections were incubated in PBS containing 0.5% Triton X-100 and 10% normal goat serum for 1 h at room temperature, following by incubation with rat monoclonal anti-BrdU (1:400; Abcam, Cambridge, United Kingdom) at 4°C overnight. After several PBS rinses, sections were incubated with Alexa Fluor 594 donkey anti-rat IgG (1:200; Invitrogen, Carlsbad, CA, United States).
An experimenter (L-cY) coded all slides from the experiments before quantitative analysis. All BrdU-labeled cells in the DG of injured hemisphere were counted in each section by another experimenter (D-qS) blinded to the study coding. The total number of BrdU-labeled cells per section was determined and multiplied by 10 to obtain the total number of cells per DG using fluorescence confocal microscopy (EX61; Olympus, Tokyo, Japan).

The following antibodies were used for immunostaining, or western blotting: mouse monoclonal anti-PLZF (Santa Cruz Biotech, Cat# sc-28319), rabbit polyclonal anti-PLZF (Santa Cruz Biotech, Cat# sc-22839), mouse monoclonal anti-γ-H2A (Millipore, Cat# 05-636), rabbit monoclonal anti-Cyclin D1 (Cell Signaling Technology, Cat# 2978), rat monoclonal anti-BrdU (Abcam Cat# ab6326), rabbit polyclonal anti-GDNF (Abcam, Cat# ab18956), rabbit polyclonal to anti-CDKN2AIP (Abcam, Cat# ab140519), rabbit polyclonal anti-MVH (Abcam, Cat# ab13840), rabbit monoclonal anti-Wilms Tumor Protein (Abcam, Cat# ab89901), mouse monoclonal anti-β-Catenin (BD Biosciences, Cat# 610154), goat Polyclonal anti-c-kit (R&D Systems, Cat# AF332-SP).

Frozen 30-μm-thick sections were incubated in blocking solution (10% normal donkey serum or 10% normal goat serum and 0.3% Triton X-100 in PBS, pH 7.5) for 2 h at room temperature (RT) and then incubated with primary antibodies in 5% serum and PBS for 24 h at 4°C. The sections were subsequently incubated for 3 h at RT with fluorophore-conjugated secondary antibodies. To prepare the sections for BrdU staining, they were incubated in 2 N HCl for 0.5 h at 37°C.
Rat monoclonal anti-BrdU (1:100; Abcam, UK) primary antibodies were used to mark proliferating cells, mouse monoclonal anti-Nestin (1:100; BD Pharmingen, USA) primary antibodies were used as a marker for NSCs, and mouse monoclonal anti-NeuN (1:100; Chemicon, USA) primary antibodies were used to label mature neurons. The following secondary antibodies were used: Alexa Fluor 594-labeled donkey anti-rat IgG and Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200 for both; Invitrogen, USA).
The stained slides were dehydrated, cover-slipped in anti-quenching agent (p-phenylenediamine, PPD) and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan). The number of positive cells was counted in a blinded manner in the ipsilateral SGZ using 20X and 40X objectives in OLYMPUS FV10-ASW Viewer.