May 26, 2018
Melanophore response assay in zebrafish
- Caio Maximino1,
- Dainara Pereira dos Santos Souza1
- 1Universidade Federal do Sul e Sudeste do Pará
- Fish behavior and physiology
Protocol Citation: Caio Maximino, Dainara Pereira dos Santos Souza 2018. Melanophore response assay in zebrafish. protocols.io https://dx.doi.org/10.17504/protocols.io.qf7dtrn
Manuscript citation:
Maximino C, Lima MG, Oliveira KR, Picanço-Diniz DL, Herculano AM (2011). Adenosine A1, but not A2, receptor blockade increases anxiety and arousal in Zebrafish. Basic Clin Pharmacol Toxicol 109: 203-207. doi: 10.1111/j.1742-7843.2011.00710.x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 25, 2018
Last Modified: May 26, 2018
Protocol Integer ID: 12511
Keywords: Zebrafish, Melanophore response, Fish physiology
Abstract
Melanophores (also called melanocytes) are chromatophores (pigment-containing epithelial cells) that contain black or dark brown pigment granules. The aggregation or dispersal of these cells is under hormonal or nervous system control; these responses permit the animal to display rapid variations in color in response to environmental stimuli, including background color and stressful stimuli. This protocol was developed at Laboratório de Neurociências e Comportamento "Frederico Guilherme Graeff", at the Universidade Federal do Sul e Sudeste do Pará (UNIFESSPA), Marabá/PA, Brazil, to assess melanophore aggregation or disperal responses in live, anesthetized adult zebrafish. The protocol is intended to be easily applied, reproducible, and of low cost.
Materials
MATERIALS
20 mg EugenolbiorbytCatalog #orb104769
Glass Petri dishes 90 x 15 cm
Dissection microscope
STEP MATERIALS
20 mg Methyl eugenolbiorbytCatalog #orb105046
Glass Petri dishes 90 x 15 cm
Dissection microscope
20 mg Methyl eugenolbiorbytCatalog #orb105046
Glass Petri dishes 90 x 15 cm
Dissection microscope
Protocol materials
20 mg Methyl eugenolbiorbytCatalog #orb105046
Glass Petri dishes 90 x 15 cm
20 mg Methyl eugenolbiorbytCatalog #orb105046
20 mg EugenolbiorbytCatalog #orb104769
Glass Petri dishes 90 x 15 cm
Glass Petri dishes 90 x 15 cm
Dissection microscope
Dissection microscope
Dissection microscope
20 mg Methyl eugenolbiorbytCatalog #orb105046
Glass Petri dishes 90 x 15 cm
Dissection microscope
Anesthetize fish by immersion in eugenol (60-100 ppm) in a 50 ml beaker.
60 Parts per Million (PPM)
20 mg Methyl eugenolbiorbytCatalog #orb105046
After anesthesia, quickly remove the animal from the solution and transfer it to a glass Petri dish (90 x 15 cm) under a dissection microscope. The Petri dish should contain enough eugenol solution to cover the animal’s body; the eugenol solution will keep the animal alive.
Glass Petri dishes 90 x 15 cm
Dissection microscope
Using tweezers, position the animal so that it lies on its side, and the dorsal portion of its body is visible under the microscope.
Take a still photo of the image. Export the file in a format that is readable by ImageJ (https://imagej.net/Importing_Image_Files)
Open the image file in FIJI ImageJ
Software
(FIJI Is Just) ImageJ
NAME
Xubuntu 16.04
OS
Curtis Rueden
DEVELOPER
SOURCE LINK
Convert the file to 8-bit (Image > Type > 8-bit)
Use the Auto Local Threshold tool to transform the image into black and white (Image > Adjust > Auto Local Threshold). The Phansalkar method for thresholding should be used, with Radius 15, both parameters set to 0; the “White objects of black background” box should be ticked.
Note
This step is crucial for reproducibility; thresholding images by using the "Threshold" tool and subjective parameters can lead to irreproducible results.
Using the polygon tool, make a selection involving the majority of melanophores in the third strip (from most dorsal to most ventral). Crop the image (Image > Crop)
Apply the Analyze Particles plugin (Analyze > Analyze Particles), restricting particles with sizes between 20-1000 pixels², and circularity from 0.0 to 1.0. In the dropdown menu “Show”, select “Outlines”. Tick the boxes “Summarize”, “Exclude on edges”, and “In situ show”; untick the other boxes, and click OK.
The summary output will display the number of particles (melanophores) detected, total area covered by melanophores (in pixels²), average melanophore size, and percentage of the image area that is covered by melanophores (in %).
Repeat the protocol for each image. If using still photos taken from the same animal, data should be summarized as average value per animal before statistical analysis to prevent pseudoreplication.