TITLE: Quantification of thiobarbituric acid reactive species (TBARS) optimized for zebrafish brain tissue
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani , Ana Herrmann, Angelo Piato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Zebrafish are increasingly used as a model animal in neuroscience research. Here we describe our protocol to quantify thiobarbituric acid reactive species (TBARS) in zebrafish brain tissue. TBARS levels are indicative of lipid peroxidation. </div></div>

[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in the quantification of thiobarbituric acid reactive species (TBARS) in the samples;
?. [Preparing the reagents]
Trichloroacetic acid (TCA) 20%  + Thiobarbituric acid (TBA) 0.5%: this reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;    1.1.1 Weigh  of TCA in a beaker of appropriate size;    1.1.2 Dissolve the TCA with  of ultrapure water;    1.1.3 Weigh carefully  of TBA in a piece of aluminum foil;    1.1.4 Add the TBA to the solution of water + TCA. Use a heating plate to help dissolve the TBA in the solution;    1.1.5 Transfer your solution to a  volumetric flask;    1.1.6 Using ultrapure water, complete the solution's volume to reach ;
2 g
8 mL
0.05 g
10 mL
10 mL
?. [Preparing the reagents]
Malondialdehyde (MDA): This reagent is volatile, be careful to maintain the storing flask closed;    Preparing a stock solution :      1.2.1 Weigh carefully  of MDA in a piece of aluminum foil;    1.2.2 Transfer the MDA to a beaker of appropriate size;    1.2.3 Add, slowly,  of absolute ethanol to the beaker to dissolve the salt;    1.2.4 Store the solution in an amber flask of appropriate size at ;    1.2.5 Each time you proceed with this thiobarbituric acid reactive species quantification method, prepare a   sample solution from your stock solution following the calculation below;C1 x V1 = C2 x V2x V1 =  x   V1 =  of the stock solution ()    1.2.6 Using a micropipette, collect  of the stock solution and mix it to  of absolute ethanol;    1.2.7 This diluted solution should be prepared on the day of the biochemical assay, it should not be stored for later use;
0.00627 g
10 mL
8 °C
5 mL
0.05 mL
50
50
4950
?. [Incubation of the samples + standard curve]
To optimize the reaction that forms the thiobarbituric acid reactive species, an incubation step is needed. Tissue sample collection and preparation are described elsewhere;
?. [Incubation of the samples + standard curve]
?. [Incubation of the samples + standard curve]
Prepare   heat resistant microtubes, to be used to store the samples, with the correct information. The number of microtubes depends on the number of samples and points of the standard curve;
1.5 mL
?. [Incubation of the samples + standard curve]
For each point of the standard curve, fill the plastic microtubes as described below. You should provide duplicates or triplicates of each point of the curve to make your quantification more precise. Using a micropipette fill the tubes in this order: MDA solution, ultrapure water, and TCA + TBA solution (mixing the solution with the pipette tip to homogenize the content);  ABCD1Point of the curveMDA (20 nmol/mL) (µL)Ultrapure water (µL)TCA 2% + TBA 0.05% solution (µL)2Control (0 nmol of MDA)01001503Point 1 (0.4 nmol of MDA)20801504Point 2 (0.8 nmol of MDA)40601505Point 3 (1.2 nmol of MDA)60401506Point 4 (1.6 nmol of MDA)80201507Point 5 (2.0 nmol of MDA)1000150
ABCD1Point of the curveMDA (20 nmol/mL) (µL)Ultrapure water (µL)TCA 2% + TBA 0.05% solution (µL)2Control (0 nmol of MDA)01001503Point 1 (0.4 nmol of MDA)20801504Point 2 (0.8 nmol of MDA)40601505Point 3 (1.2 nmol of MDA)60401506Point 4 (1.6 nmol of MDA)80201507Point 5 (2.0 nmol of MDA)1000150
?. [Incubation of the samples + standard curve]
Before preparing your samples for incubation, you must calculate the sample volume that corresponds to of proteins. This calculation is based on the Bradford method described elsewhere;    2.3.1 To estimate the volume of the sample corresponding to  of proteins, divide the amount of protein needed () by the total amount of proteins in the sample quantified by the Bradford method (example below);Volume of the sample needed for the assay (µL) = 50 µg / total amount of proteins in the sample µg/µL
50 µg
50 µg
50 µg
?. [Incubation of the samples + standard curve]
For each tissue sample, fill the plastic microtubes as described below. You should provide duplicates or triplicates of each sample to make your quantification more precise. Using a micropipette fill the tubes in this order: sample, ultrapure water, and TCA + TBA solution (mixing the solution with the pipette tip to homogenize the content). The water volume depends on the volume of the sample. All microtubes should have a final volume of , so water is used so that every solution reaches this volume (e.g.  of the sample +  of the TCA + TBA solution +  of water);  ABCD1MicrotubesSample (µL)Ultrapure water (µL)TCA 2% + TBA 0.05% solution (µL)2Control01001503SampleDepends on the volume of the sample corresponding to 50 µg  of proteins.Depends on the volume of the sample. Volume needed for the final solution in the tube to reach 250 µL.150
250
50
150
50
ABCD1MicrotubesSample (µL)Ultrapure water (µL)TCA 2% + TBA 0.05% solution (µL)2Control01001503SampleDepends on the volume of the sample corresponding to 50 µg  of proteins.Depends on the volume of the sample. Volume needed for the final solution in the tube to reach 250 µL.150
?. [Incubation of the samples + standard curve]
All microtubes should be correctly closed and sealed with adhesive tape to avoid that the microtubes open during the incubation period;
?. [Incubation of the samples + standard curve]
Incubate all your samples at  for  using a dry bath;
100 °C
?. [Calculating data and determinig results]
The calculations are based on the MDA curve (similar to the calculation of the protein curve described in other protocols);
?. [Calculating data and determinig results]
Calculate the correction factor for your MDA standard curve;    4.1.1 Correction factor (CF): Subtract the absorbance value of the control point of the curve from the mean absorbance of the point you are calculating. Divide the concentration of MDA of the well/point of the curve by the resulting value from the subtraction before;CF = _____[  ] MDA (nmol)____           Mean Abs – Control Abs    4.1.2 Mean correction factor (MCF): The mean correction factor is calculated by the arithmetic mean of the correction factors for each point of the curve;MCF = ∑Correction factors / Count of correction factorsor MCF  = _FC1+ FC2 + FC3 + FC4 + FC5_        5(number of factors)
?. [Calculating data and determinig results]
The amount of thiobarbituric acid reactive species in your samples is calculated by multiplying the corrected mean absorbance of your sample to the mean correction factor calculated above;TBARS = (Mean absorbance of the sample - Mean absorbance of the control sample) x MCF
?. [Calculating data and determinig results]
Results should be expressed as nmol of TBARS/mg of protein.  To do so, divide the result obtained above for the amount of thiobarbituric acid reactive species in your sample by the amount of protein that you used of your sample   ( or   in this protocol).nmol TBARS/mg protein = nmol TBARS on the sample / 0.05 mg of proteins
50 µg
0.05 mg
?. [Reading your samples]
Prepare to read the absorbance of your samples in a microplate reader;
?. [Reading your samples]
Use a conventional 96-well microplate to run your samples. Before start pipetting, each well of the microplate should be marked for sample identification.. Standard curve samples and tissue samples can be read on the same plate. Transfer  of the content of each microtube to it's corresponding well in the microplate;
200
?. [Reading your samples]
Read the absorbance of the samples at  in a microplate reader;
532