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Table 1 RNAscope protocol for zebrafish embryos at a glance

From: Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos

Step Solution Temperature Time
1. Drying RT 30 min
2. Digestion 2 drops of Pretreat 3 RT 20 min
3. Stop digestion PBT 1 ml RT Quick 3×
4. Probe hybridization Channel 1: Channel 2: Channel 3 (50:1:1) 50–100 μl/tube 40°C O/N
5. Wash 0.2× SSCT 1 ml RT 3 × 15 min
6. Post-fixation 4% PFA in PBS 1 ml RT 10 min
7. Wash 0.2× SSCT 1 ml RT 3 × 15 min
8. Pre-amplifier hybridization 2 drops of Amp1 40°C 30 min
9. Wash 0.2× SSCT 1 ml RT 3 × 15 min
10. Signal enhancement 2 drops of Amp2 40°C 15 min
11. Wash 0.2× SSCT 1 ml RT 3 × 15 min
12. Amplifier hybridization 2 drops of Amp3 40°C 30 min
13. Wash 0.2× SSCT 1 ml RT 3 × 15 min
14. Label 2 drops of Amp4 40°C 15 min
15. Wash 0.2× SSCT 1 ml RT 3 × 15 min
16. Counter stain 2 drops of DAPI or Hoechst: SSCT (1:10000) 4°C O/N
17. Mounting 1% LMP RT 5–10 min
18. Microscopy
  1. DAPI, 4’ ,6-diamidino-2-phenylindole; LMP, low melting point agarose; O/N, overnight; PBS, phosphate-buffered saline; PBT, PBS buffer + 0.01% Tween-20; PFA, paraformaldehyde; RT, room temperature; SSCT, saline-sodium citrate buffer + 0.01% Tween-20.