5-Fluorouridine

This plan only provides a guide, please modify it to meet your specific needs.

1. Solution preparation

(1) Working solution: Dissolve 5-Fluorouridine in the experimental buffer and prepare the working solution of the required concentration.

Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.

2. 5-Fluorouridine in vivo labeling^[1](Source literature, for reference only)

(1) Inject 5-Fluorouridine intraperitoneally into mice at a dose of 10μL/g. 0.4M 5-Fluorouridine stock solution was dissolved in 0.9% saline.

(2) Euthanize the mice 45min after injection and fix them by perfusion with 3.7% paraformaldehyde in HPEM buffer (2×HPEM: 60mM Hepes; 130mM Pipes; 20mM EGTA; 4mM MgCl₂·6H₂O) containing 0.5% Triton X for 10min.

(3) The brain was removed, fixed with perfusion buffer for 1h, and washed in HPEM.

(4) Mechanical hippocampal granule cell dissociation was performed.

(5) The cells were treated with mouse monoclonal anti-BrdU at 37°C for 1h.

(6) After washing with PBS solution containing 0.1% Tween20, the cells were incubated with FITC-conjugated secondary antibody for 45min at room temperature.

(7) After washing with PBS, the slides were sealed and the incorporation of 5-Fluorouridine into RNA was detected under a fluorescence microscope.

3. 5-Fluorouridine labeling in vitro^[2] (Source literature, for reference only)

(1) The cells were pulse-labeled with 2mM 5-Fluorouridine for 20min.

(2) The cells were fixed with 2% formaldehyde for 10min, permeabilized with 0.5% Triton X-100 for 10min, and then blocked with PBS solution containing 0.1% Tween20 for 1h.

(3) Incubate with BrdU antibody at 4°C overnight.

(4) After washing with PBS, incubate the cells with FITC-conjugated secondary antibody at room temperature for 1h.

(5) After washing with PBS, treat the cells with DAPI and detect the incorporation of 5-Fluorouridine into RNA under a fluorescence microscope.

Note: For your safety and health, please wear a lab coat and disposable gloves when operating.

References:

[1] Puente-Bedia A, Berciano M T, Tapia O, et al. Nuclear reorganization in hippocampal granule cell neurons from a mouse model of Down syndrome: changes in chromatin configuration, nucleoli and Cajal bodies[J]. International Journal of Molecular Sciences, 2021, 22(3): 1259.

[2]Awasthi S, Verma M, Mahesh A, et al. DDX49 is an RNA helicase that affects translation by regulating mRNA export and the levels of pre-ribosomal RNA[J]. Nucleic Acids Research, 2018, 46(12): 6304-6317.