@Babajimi Ogunleye

Alternative Method: Bath Loading with AM Ester Dyes

What You’ll Need:

Calcium indicator dye: e.g. Fluo-4 AM, Cal-520 AM, or Rhod-2 AM (choose based on your imaging setup: excitation/emission wavelengths)

Pluronic F-127 (helps dissolve the dye)

DMSO (dimethyl sulfoxide, for initial dye dilution)

E3 embryo medium (or your regular zebrafish medium)

4 dpf zebrafish larvae

Protocol Overview (for Fluo-4 AM, adjust for others as needed):

1. Prepare dye stock:

Dissolve the AM dye (e.g. Fluo-4 AM) in DMSO with 20% Pluronic F-127.

Example: 50 µg Fluo-4 AM in 50 µL DMSO + Pluronic F-127.

2. Prepare loading solution:

Dilute your dye stock into E3 medium (final dye concentration usually ~5–10 µM).

Optional: Add 0.02% Tricaine to anesthetize the larvae.

3. Incubate larvae:

Place 4 dpf zebrafish larvae in the dye solution.

Incubate at 28.5°C in the dark for 30–60 minutes.

4. Wash thoroughly:

Wash the larvae 3–4 times with fresh E3 medium to remove excess extracellular dye.

Allow recovery for 15–30 minutes before imaging.

🔎 Tips:

Dechorionated larvae (which they usually are at 4 dpf) are better for dye uptake.

This method works well for global calcium imaging, especially in superficial tissues like the brain, spinal cord, or muscles.

For deeper tissue, uptake can be limited—sometimes electroporation or targeted delivery may be needed.

Alternatives If Bath Loading Fails:

If bath loading doesn't give strong enough signal or specificity:

Electroporation of dye into specific tissues

Liposomal delivery

@ Kudakwashe Mushavanhu thank you so very much. id like to confirm if there is any paper i can read or cite for my write up?