TITLE: Extracellular fluid extraction in zebrafish brain tissue and samples
AUTHORS: Caio Maximino

[DESCRIPTION]
This protocol is used to extract the contents of the extracellular fluid of the adult zebrafish brain, allowing quantification of analytes such as neurotransmitter (e.g., Maximino et al., 2013) or proteins (e.g., Pradel et al., 1999).

[STEPS]
SECTION: Euthanasia and dissection
3. Sacrifice animals in ice-cold water.
 

SECTION: ECF extraction
5. In a 1.5 mL microtube filled with extraction fluid, add one brain (or fraction). Keep the microtube on ice or on the fridge, maintaining the temperature at 4 ºC. Incubate for 30 min.
4 °C 
30 min 

SECTION: Quantification
6. Samples can be analyzed using sensitive techniques, such as HPLC, to assay neurotransmitter content. 
 

SECTION: Euthanasia and dissection
3.1. Add ice to a beaker filled with at least 1 L system water. Measure temperature so that it falls between 0 ºC and 4 ºC.4 °C 
Add a tea strainer above the ice layer, where individual animals will be placed. The use of the strainer allows cooling the water while avoiding direct contact of the fish body with the ice.

SECTION: Reagent preparation
1. Prepare the extraction fluid. The recipe below is good for 1 L.

 


SECTION: Reagent preparation
1.1. To make 1 L Tris buffer
Add 4.44 g  to 1 L double distilled water
Add 2.65 g   
Check if pH 8.0 . If not, adjust with acid or base


SECTION: Reagent preparation
1.2. To the Tris buffer, add 5.2596 g   

SECTION: Reagent preparation
1.3. Add 0.27745 g   

SECTION: Reagent preparation
1.4. Add 0.30733 g  

SECTION: Reagent preparation
1.5. Adjust pH to pH 7.4 

SECTION: Reagent preparation
2. Prepare dissection solution (artificial cerebrospinal fluid). The recipe below is good for 1 L.
 

SECTION: Reagent preparation
2.1. Add 7.65564 g   to 1 L double distilled water
Add 0.1491026 g   
Add 0.1701 g   
Add 0.240732 g  
Add 1.80156 g   
Add 0.27745 g   
Add 1.68014 g   












SECTION: Reagent preparation
2.2. Keep dissection solution on the fridge (4 ºC) for a maximum of 30 days before use. 
SECTION: Euthanasia and dissection
3.2. Transfer animals individually to the strainer. Maintain animals for at least 5 min in contact with the cold water. If animals show any sign of activity, allow for more time in the ice. 
SECTION: Euthanasia and dissection
4. Decapitate and dissect the animal.
SECTION: Euthanasia and dissection
4.1. Transfer the animal to a Petri dish filled with dissection fluid.
 

SECTION: Euthanasia and dissection
4.2. Decapitate the animal by cutting cleanly through the pectoral girdle with dissection scissors. The cut 
should be made immediately anterior to the articulation of the pectoral fin with the girdle, severing the heart.
 

SECTION: Euthanasia and dissection
4.3. Using the dissecting scissors, remove the skin and bones from the head, exposing the brain. To avoid damaging the forebrain, start dissection at the level of the junction between medulla and spinal cord. Gently raise the medulla with an insulin needle and cut the ventral roots of the cranial nerves using microdissection pincers.
SECTION: Euthanasia and dissection
4.4. The tissue sample can be assessed as a whole, or microdissected into forebrain, midbrain, and hindbrain.
SECTION: Quantification
6.1. The remaining tissue can be used to assay other analytes, such as second messenger levels.