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

Table 1 RNAscope protocol for zebrafish embryos at a glance

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	–	–	–

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.

ISSN: 1741-7007