I'm not an expert in zebrafish, but from my experience in a developmental biology lab during my BA, where we utilized zebrafish as model organisms, I can provide a general outline for measuring melanin content in zebrafish embryos/larvae using the absorbance method. Here's a basic protocol:

Materials:

Zebrafish embryos/larvae

Phosphate-buffered saline (PBS)

Lysis buffer (e.g., 1% Triton X-100, 0.1 M PBS)

Hydrochloric acid (HCl)

Isopropanol

Sodium hydroxide (NaOH)

3-Isobutyl-1-methylxanthine (IBMX)

Ascorbic acid

Dopachrome standard

Microplate reader

Procedure:

Sample Preparation:

Collect zebrafish embryos/larvae.

Wash them with PBS to remove debris and excess pigments.

Tissue Homogenization:

Homogenize the embryos/larvae in lysis buffer.

Centrifuge the homogenate to obtain a clear supernatant.

Melanin Extraction:

Add HCl to the supernatant to hydrolyze melanin.

Extract melanin with isopropanol.

Centrifuge to obtain the melanin-containing supernatant.

Alkaline Hydrolysis:

Add NaOH to the melanin-containing supernatant.

Incubate to alkaline hydrolyze melanin.

Color Development:

Add IBMX and ascorbic acid to the hydrolyzed sample.

Incubate to allow for color development.

Absorbance Measurement:

Measure the absorbance at a specific wavelength using a microplate reader.

Use a blank control (no sample) for background subtraction.

Melanin Quantification:

Create a standard curve using a known concentration of dopachrome standard.

Calculate the melanin concentration in the samples based on the standard curve.

Data Analysis:

Normalize melanin content to protein concentration if needed.

Perform statistical analysis if comparing between different experimental groups.

Notes:

Ensure proper controls and replicates in your experiment.

Optimize parameters such as incubation time, concentrations, and wavelengths for your specific conditions.

This is a standard protocol; for more detailed information, refer to this article

doi: 10.3389/fphar.2018.00265

Moreover, engaging in discussions with experts in the field could yield additional insights. Best of luck with your research!