mRNA In Situ Hybridization with Nick-Translated Probes

1. Cells are fixed with freshly made 4% formaldehyde in PBS, pH 7.4 for 15 min at room temperature. All solutions should be made in Molecular Biology grade ultrapure water (no RNase). Wear gloves at all times and use sterile disposable pipets and tips.
2. After rinsing in PBS (3 X 10 min. each), cells are permeabilized with 0.5% Triton X-100 in 1X PBS for 10 min at 4oC.
3. Cells are then rinsed in PBS (3 X 10 min. each) and then in 2X SSC (1 X 5 min.).
4. 100 ng of nick translated probe (containing digoxigenin dUTP) and 20 ug of competitor E. coli tRNA per coverslip are dried down in a Speed Vac (Savant). This is a good starting place but you may have to titrate your specific probe.
5. 10 μl of deionized formamide is added to the dried DNA.
6. The probe and tRNA are denatured by heating for 10 min at 90oC. The probe is chilled on ice immediately.
7. 10 μl of Hybridization buffer (20% dextran sulfate + 4X SSC) is added to the denatured probe so that the final concentrations in the hybridization mixture are 5 ng/ml of probe, 1 ug/ml of E. coli tRNA, 2X SSC, and 10% dextran sulfate.
8. 20 μl of hybridization mixture/probe is placed onto each coverslip.
9. Coverslips are inverted onto a slide and sealed with rubber cement and incubated in a humid chamber for 16 hrs. at 37oC.
10. After rinsing in 2X SSC/50% formamide at 37oC, 2X SSC and 1X SSC at room temperature for 30 min. each, the coverslip containing cells are incubated in 4X SSC/0.25% BSA/2ug/ml anti-digoxigenin antibody for 60 min. in a humid chamber at room temperature in the dark.
11. Coverslips are then rinsed in 4X SSC (1 X 15 min.) at room temperature, 4X SSC/0.1% Triton X-100 (1 X 15 min.), and 4X SSC (3 X 10 min. each).
12. Coverslips are mounted in fluorescence mounting medium.

Modified from: Jiménez-García, L. and D.L. Spector. 1993. Cell 73, 47-59.