Anti brdu clone bu 33

Active transcription sites were labelled by incorporation of 5′-fluorouridine (5′-FU) into nascent RNA as previously described [60 (link)]. The mice were given an intraperitoneal injection of 5-FU (Sigma, Darmstadt, Germany) at a dose of 10 μL/g of a stock solution of 0.4 M 5-FU in 0.9% saline. The mice were euthanized 45 min post-injection and fixed by perfusion with 3.7% paraformaldehyde in HPEM buffer (2× HPEM: Hepes, 60 mM; Pipes, 130 mM; EGTA, 20 mM; and MgCl₂·6H₂O, 4 mM) containing 0.5% Triton X for 10 min. Their brains were removed, post-fixed with a perfusion buffer for 1 h and washed in HPEM. Then, mechanical GCs dissociation was performed as described above. The incorporation of 5′-FU into nascent RNA was detected with mouse monoclonal anti-BrdU (Clone BU-33, Sigma, Darmstadt, Germany) for 1 h at 37 °C. Then, the samples were washed with 0.01% Tween 20 in PBS, incubated for 45 min with an anti-mouse FITC-conjugated secondary antibody, washed in PBS and mounted with Vectashield. Quantification of the intensity of 5′-FU fluorescence was determined within the nuclear perimeter in at least 100 GCs from three animals of each karyotype.