Whole mount in situ hybridization

P-EV N 000016

Optimized version by EV

RNAscope® Assay on Whole Zebrafish Embryos

Note: This protocol is designed to be used in conjunction with the RNAscope® Multiplex Fluorescent Reagent Kit v2 (Cat. No.  323100, acdbio.com) 

Part 1: Sample Preparation

Fix the Embryos

NOTE: Use sterile solutions. Use UltraPure RNAse free water for 1xWash buffer and 1xPBST preparation. 

1. Collect zebrafish embryos at the desired developmental stages and remove the chorions.

2. Place the embryos into 1.5ml tubes (2-3 fish per tube for 1-5dpf, 1 fish for 14dpf). All steps will be performed in 1.5 tubes. 

3. Fix the embryos using 1.5 mL per tube of fresh 4%PFA in 1xPBS at ROOM TEMPERATURE (RT) for 24 HRS.

4. Remove the fixative solution. Wash the embryos using 1 mL per tube of 1X PBS + 0.1% Tween 20 (PBST) at RT for 10 MIN. 

Dehydrate and Store the Embryos

1. Replace 1xPBST with 1 mL of 30% methanol in 1X PBST. Incubate at RT for 10 MIN.
2. Replace 25% methanol with 1 mL of 50% methanol in 1X PBST. Incubate at RT for 10 MIN.
3. Replace 50% methanol with 1 mL of 70% methanol in 1X PBST. Incubate at RT for 10 MIN.
4. Replace 75% methanol with 1 mL of 100% methanol. Incubate at RT for 10 MIN.

NOTE: Store the embryos in 100% methanol at –20ºC for up to two months. Make sure the embryos do not dry out.

Part 2: Sample Pretreatment

Rehydrate and Permeabilize the Embryos

NOTE: All steps will be performed in 1.5 tubes.

1. Incubate embryos in 1ml of 75% methanol in 1X PBST at RT for 10 MIN.
2. Incubate embryos in 1ml of 50% methanol in 1X PBST at RT for 10 MIN.
3. Incubate embryos in 1ml of 25% methanol in 1X PBST at RT for 10 MIN.
4. Incubate embryos in 1ml of PBST + 1% BSA at RT for 10 MIN.

Apply RNAscope® Target Retrieval

1.  Preheat the 1x Target Retrieval solution in a heating block to 100°C.
2. Carefully replace the PBST+1%BSA with the heated 1X Target Retrieval solution. Incubate at 100°C for 10 MIN (24-48hpf), or 15 MIN (3-14dpf).
3. Immediately replace the Target Retrieval solution with 1ml PBST + 1% BSA. Incubate for 1 MIN.
4. Wash your probes with 100% methanol for 1 MIN. 
5. Remove 100% methanol. Wash the embryos carefully by slowly adding 1 mL PBST + 1% BSA one drop at a time.
    

IMPORTANT! The embryos may stick to the side of the tube when PBST + 1% BSA is added. If this occurs, replace PBST + 1% BSA with 100% methanol and repeat steps 6 and 7.

Apply RNAscope® Protease Plus

1. Carefully remove as much of the PBST + 1% BSA wash as possible without letting the embryos dry. 

2. Add 1 drops of Protease Plus to each tube, and incubate at 40ºC for 5–60 MIN depending on the age of the embryos:

• 2 minutes for 24 hpf
• 5 minutes for 48 hpf
• 5-10 minutes for 72hpf
• 15-20 minutes for 14dpf

3. Replace Protease Plus with 1 drops of Probe Diluent.

4. Remove Probe Diluent before continuing with the assay.

Part 3: RNAscope® Assay

Probe Hybridization and Staining

NOTE: Be gentle when adding all solutions, embryos are very fragile.

Continue with the RNAscope® 2.5 HD or V2 fluorescent assay steps from target probe hybridization to counterstaining using Part 4 of the 323100 protocol.

Make the following modifications:

• Do not forget to prewarm your probes before diluting them (see page 30: Prepare probes)
• Perform all hybridization steps in a 40ºC water bath (volumes: 1 drop per tube, etc).
• Perform all wash steps twice using 1ml of 1X Wash Buffer for 5 MIN each time with gentle (!!!) shaking.
• Opal520, cy3, cy5 dilution: 1:750. Keep in dark!

• OPTIONAL: At the end of the RNAscope® assay, clear the embryos with CUBIC reagent by removing the wash buffer and adding 200 μL of CUBIC reagent (see below). Store at RT.