Rat anti brdu antibody

For cell cycle analysis, HUES6 cells were individualized using accutase 1:4 in PBS and fixed in 70% ethanol overnight. For BrdU detection, cells were incubated with 10 μM BrdU for 30 minutes before collection. After incubation with 0.5 mg/ml pepsin cells were treated with 2 N HCl for 20 minutes, washed and incubated with a rat-antiBrdU antibody (AbD serotec) for 45 minutes followed by incubation with goat anti-rat-FITC secondary antibody (Southern Biotech) for 30 minutes. Cells were then resuspended in PBS containing 50 μg/ml of propidium iodide and 0.5 mg/ml of RNaseA. Flow cytometry analyses were conducted using a LSRII cytometer (BD Bioscience) and data was analyzed using FlowJo software.

To understand whether AOB neurons recruited for transmitter respecification represent newly born cells or pre-existing neurons, cells proliferating during kin/non-kin conditioning were identified by BrdU (Sigma) labeling. Stage 39 larvae were exposed to BrdU by immersion in 4 mg/ml in 10% MMR for 24 hr. Specimens (stage 42 and 45) were fixed and sucrose-cryoprotected. 10 μm cryostat sections were treated for 20 min in 2 M hydrochloric acid (HCl; Fisher Scientific) for antigen retrieval and then washed and incubated with rat anti-BrdU antibody (AbD Serotec) overnight.

DNA fiber assays to analyze replication fork progression and origin firing were essentially carried out as described previously^{38 (link)}. Cells were first incubated with 4-OHT for 6 h and afterwards with 5-chloro-2-deoxyuridine (CldU, 25 μM) for 20 min, followed by 5-iodo-2-deoxyuridine (IdU, 25 μM; both from Sigma-Aldrich) for 1 h. DNA fibers were spread on glass slides. After acid treatment, CldU- and IdU-labeled tracts were detected by 1 h incubation at 20 °C with rat anti-BrdU antibody (dilution 1:400 detects BrdU and CldU; AbD Serotec) and mouse anti-BrdU antibody (1:150, detects BrdU and IdU; Becton Dickinson). Slides were fixed in 4 % paraformaldehyde/PBS and incubated for 2 h at 20 °C with Alexa Fluor 555-conjugated goat anti-rat antibody (dilution 1:150) or Alexa Fluor 488-conjugated goat anti-mouse antibody (dilution 1:150; both from Molecular Probes/Thermofisher). Fiber images were acquired by fluorescence microscopy using the Axio Scope A1 running with the microscope software ZEN (both from Zeiss) for image acquisition and processing. For analysis of fiber images, the imaging software ImageJ was used. Statistical analysis of replication fork progression was performed using the two-tailed, unpaired t-test with additional Welch’s correction in Prism5.0 due to unequal variances between samples.