Induction of recombination by 4-OH Tamoxifen intraperitoneal injection in adult zebrafish

This is a step-by-step protocol for the induction of recombination by 4-OH Tamoxifen intraperitoneal injection in adult zebrafish.

1) Preparation of 4-OH Tamoxifen stock

It’s best to use freshly dissolved 4-OH Tamoxifen. Aliquots of dissolved solutions kept at -20°C are stable for up to 3 months. It is not recommended to use older dissolved aliquots.

• Use (Z)-4-Hydroxytamoxifen (4-OHT) which contains > 98 % Z-isomer
• The final concentration of the stock should be 10 mM, the solvent is 100% EtOH.
• Prepare 20 µl aliquots. Store aliquots at -20°C.
  • It is best to use PCR tubes. In larger tubes, the injection needle might not reach the bottom. 
• Use aliquots of 4-OH Tam dissolved in EtOH only once and DO NOT re-freeze an aliquot.

2) 4-OH Tamoxifen intraperitoneal injection in adult zebrafish

• Injection volume:10 µl
• Injection concentration: 3.4 mM 
   

Injection regime

• How many injections are needed for efficient recombination and in which time-frame they should be applied will depend on the CreERT2 line.
• Four injections appear to be sufficient for most lines.
• Fish can tolerate consecutive injections every 24 hours for few days, but a day of rest every 2-3 injections is recommended.

Preparation of the injection mix

• It is important to inject the diluted 4-OHT  within 10 min, otherwise the solution turns clear which is not good. Thus, prepare everything for injection before you dilute the 4-OHT stock.
• Make sure there are no precipitates when using an aliquot.
• Prepare the 3.4 mM injection mix by diluting with PBS.
• The solution will turn milky, which is normal. If clear solution appears and precipitate collets at the bottom of the tube, it probably means the 4-OHT has precipitated. In this case, vortex!

I.P. injection

• Use a 10 µl or 100 µl syringe with 33 GA needles. 
  Using 10 or 100 µl syringe depends on personal preference. 
• Use a soft sponge with an incision as fish holder. Soak the sponge with system water, and put it into a 60 mm Petri dish.
• Anaesthetize fish with 625 µM tricaine.
• Clamp the anaesthetized fish belly-up into the incision. The tail should point towards the hand with which one is injecting.
• Prick into the midline between the pelvic fins. After injection of 10 µl, carefully withdraw the needle.
• Transfer the fish back to its tank.

Material

• (Z)-4-Hydroxytamoxifen
• 100 % Ethanol
• PBS
• tricaine
• fish holder: sponge with incision
• 10 µl or 100 µl syringe
• injection needles (e.g. World Precision Instruments, NanoFil NF33BV 33GA, beveled needle, NF33BV-2)