Alexa fluor 488 antibody

To measure DNA synthesis, B cell cultures were stimulated for 28 hours, pulsed with 10 mM of EdU (5-ethynyl-2^’ -deoxyuridine) for 15 min at 37°C and stained using the Click-IT EdU Alexa Fluor 488/647 Flow Cytometry Assay Kit according to the manufacturer’s specifications (ThermoFisher). DNA content was measured with DAPI or Propidium Iodide. Samples were acquired on a FACSCantoll (BD biosciences) or Accuri C6 (BD biosciences). To measure H3S10p, cells were fixed overnight with 70% ethanol, permeabilized with 0.25% Triton X-100 for 10 minutes, washed in PBS, incubated for 3 hours with H3S10p antibody (1:200, Millipore), washed, stained with anti-rabbit Alexa Fluor 488 antibody (1:2000, Thermofisher). Propidium iodide + RNAase A was added before FACS analysis on BD Accuri C6 (BD Biosciences). Data was analyzed using FlowJo, gating on live cells by FSC,SSC and single cells by FSC-H,FSC-A.

We resuspended 10 mg adult forebrain tissue (P56) in 500 µ L lysis buffer (0.5% BSA, 0.1% Triton-X, cOmplete (Roche), 1 mM DTT in PBS) and incubated it for 10 min at 4 °C. After spinning down (5 min, 500 g), the sample was resuspended in 500 µ L staining buffer (0.5% BSA in PBS). The nuclei suspension was incubated with anti-NeuN antibody (1:5,000, MAB377, Lot 2806074, EMD Millipore) for 30 min at 4 °C. After centrifugation, nuclei were resuspended in 500 µ L staining buffer (0.5% BSA in PBS) containing antimouse Alexa Fluor-488 antibody (1:1,000, A11001, Lot 1696425, Thermo Fisher Scientific). After incubating for 30 min at 4 °C, nuclei were pelleted (5 min, 500 g) and resuspended in 700 uL sort buffer (1% BSA, 1 mM EDTA in PBS). After filtration into a FACS tube, 5 uL DRAQ7 (Cell Signaling Technologies) were added and NeuN-negative nuclei were sorted using a SH800 sorter (Sony) into 5% BSA (Sigma) in PBS.

Sixty min after i.c.v. infusion, mice were euthanized with 7% chloral hydrate and perfused with saline followed by 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4). Brains were post fixed in 4% paraformaldehyde/30% sucrose in PBS at 4°C overnight. Coronal brain sections (40 μm) were cut, stored at 4°C for up to one week in PBS containing 0.02% sodium azide, washed in 0.1 M PBS, preincubated in blocking buffer (PBS + 0.2 % triton X-100 + 10% normal horse serum (NHS)) for 2 h at room temperature, and incubated with antibody to Fos (1:2000, Cell Signaling, #5348) in 2% NHS blocking buffer for 48 h at 4°C. After washing in PBS, tissue was incubated for 4 h with donkey anti-rabbit Alexa Fluor 488 antibody (1:500 in PBS + 2% NHS blocking buffer, ThermoFisher Scientific #A31556). Fos was visualized by fluorescence microscopy with Olyvia software (Olympus Life Sciences). A blinded individual counted the Fos-positive neurons using Image J software (NIH). Display images were adjusted for brightness and contrast. Brain anatomy was defined using (Franklin and Paxinos, 2007 ).
Data are reported as mean ± SEM. Significance (two-tailed p < 0.05) was determined by t-test or ANOVA followed by post hoc Holm-Sidak multiple comparison tests.