Sep 08, 2020
Quantification of thiobarbituric acid reactive species (TBARS) optimized for zebrafish brain tissue
- Adrieli Sachett1,
- Matheus Gallas-Lopes1,
- Greicy M M Conterato2,
- Radharani Benvenutti Benvenutti1,
- Ana P Herrmann1,
- Angelo Piato1
- 1Universidade Federal do Rio Grande do Sul;
- 2Universidade Federal de Santa Catarina
- Fish behavior and physiology
- LAPCOM
Protocol Citation: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti Benvenutti, Ana P Herrmann, Angelo Piato 2020. Quantification of thiobarbituric acid reactive species (TBARS) optimized for zebrafish brain tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bjp8kmrw
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: August 13, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 40416
Keywords: TBARS, Oxidative stress, Zebrafish brain tissue,
Abstract
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.
Guidelines
This protocol is intended to standardize quantification of thiobarbituric acid reactive species in zebrafish brain tissue samples. It can be adapted for other fish species.
Materials
MATERIALS
Gloves
Incubator
96 well plate
Eppendorf tubes 1.5 mL uncoloredEppendorf CentrifugeCatalog #022363204
Compact Digital Dry Bath/ Block Heater, Compact Dry Bath S, 100-240V, US plugThermo FisherCatalog #88871001
Surgical mask
Micropipette (0.5 - 10 μL)
Micropipette (100 - 1000 μL)
Multichannel pipette (5 μL; 30-300 μL)
Adhesive tape
Synergy™ HTX Multi-Mode Microplate ReaderBiotek
STEP MATERIALS
Trichloroacetic acid (TCA)Sigma – AldrichCatalog #T6399
Thiobarbituric acid (TBA)J.T. Baker
Malondialdehyde tetrabutylammonium saltSigma-aldrichCatalog #36357
EthanolMerck MilliporeCatalog #100983
EthanolMerck MilliporeCatalog #100983
Protocol materials
Synergy™ HTX Multi-Mode Microplate ReaderBiotek
EthanolMerck Millipore (EMD Millipore)Catalog #100983
96 well plate
Surgical mask
Malondialdehyde tetrabutylammonium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #36357
EthanolMerck Millipore (EMD Millipore)Catalog #100983
Eppendorf tubes 1.5 mL uncoloredEppendorfCatalog #022363204
Micropipette (0.5 - 10 μL)
Micropipette (100 - 1000 μL)
Gloves
Incubator
Trichloroacetic acid (TCA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T6399
Thiobarbituric acid (TBA)J.T. Baker
Compact Digital Dry Bath/ Block Heater, Compact Dry Bath S, 100-240V, US plugThermo FisherCatalog #88871001
Multichannel pipette (5 μL; 30-300 μL)
Adhesive tape
Thiobarbituric acid (TBA)J.T. Baker
Trichloroacetic acid (TCA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T6399
EthanolMerck Millipore (EMD Millipore)Catalog #100983
EthanolMerck Millipore (EMD Millipore)Catalog #100983
Malondialdehyde tetrabutylammonium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #36357
Safety warnings
Use personal protective equipment (including lab coat, masks, and gloves) whenever manipulating chemical and biological samples. Make sure to read all Safety Data Sheets for the reagents.
Before start
This protocol was standardized at LAPCOM (Psychopharmacology and Behavior Laboratory at UFRGS) to assess biochemical parameters in zebrafish brain tissue. Protocols you should read before proceeding with this method:
CITATION
CITATION
Preparing the reagents
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;
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 2 g of TCA in a beaker of appropriate size;
Trichloroacetic acid (TCA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T6399
1.1.2 Dissolve the TCA with 8 mL of ultrapure water;
1.1.3 Weigh carefully 0.05 g of TBA in a piece of aluminum foil;
Thiobarbituric acid (TBA)J.T. Baker
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 10 mL volumetric flask;
1.1.6 Using ultrapure water, complete the solution's volume to reach 10 mL ;
Malondialdehyde (MDA): This reagent is volatile, be careful to maintain the storing flask closed;
Preparing a stock solution 2000 nmol/mL :
1.2.1 Weigh carefully 0.00627 g of MDA in a piece of aluminum foil;
Malondialdehyde tetrabutylammonium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #36357
1.2.2 Transfer the MDA to a beaker of appropriate size;
1.2.3 Add, slowly, 10 mL of absolute ethanol to the beaker to dissolve the salt;
EthanolMerck Millipore (EMD Millipore)Catalog #100983
1.2.4 Store the solution in an amber flask of appropriate size at 8 °C ;
1.2.5 Each time you proceed with this thiobarbituric acid reactive species quantification method, prepare a 20 nmol/mL sample solution from your stock solution following the calculation below;
C1 x V1 = C2 x V2
2000 nmol/mL x V1 = 20 nmol/mL x 5 mL
V1 = 0.05 mL of the stock solution (50 µL )
1.2.6 Using a micropipette, collect 50 µL of the stock solution and mix it to 4950 µL of absolute ethanol;
EthanolMerck Millipore (EMD Millipore)Catalog #100983
1.2.7 This diluted solution should be prepared on the day of the biochemical assay, it should not be stored for later use;
Incubation of the samples + standard curve
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;
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Prepare 1.5 mL 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;
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);
| Point of the curve | MDA (20 nmol/mL) (µL) | Ultrapure water (µL) | TCA 2% + TBA 0.05% solution (µL) | |
| Control (0 nmol of MDA) | 0 | 100 | 150 | |
| Point 1 (0.4 nmol of MDA) | 20 | 80 | 150 | |
| Point 2 (0.8 nmol of MDA) | 40 | 60 | 150 | |
| Point 3 (1.2 nmol of MDA) | 60 | 40 | 150 | |
| Point 4 (1.6 nmol of MDA) | 80 | 20 | 150 | |
| Point 5 (2.0 nmol of MDA) | 100 | 0 | 150 |
The final volume in all microtubes should be 250 µL.
Before preparing your samples for incubation, you must calculate the sample volume that corresponds to 50 µg of proteins. This calculation is based on the Bradford method described elsewhere;
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2.3.1 To estimate the volume of the sample corresponding to 50 µg of proteins, divide the amount of protein needed (50 µg ) 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
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 250 µL , so water is used so that every solution reaches this volume (e.g. 50 µL of the sample + 150 µL of the TCA + TBA solution + 50 µL of water);
| Microtubes | Sample (µL) | Ultrapure water (µL) | TCA 2% + TBA 0.05% solution (µL) | |
| Control | 0 | 100 | 150 | |
| Sample | Depends 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 |
All microtubes should be correctly closed and sealed with adhesive tape to avoid that the microtubes open during the incubation period;
Incubate all your samples at 100 °C for 00:30:00 using a dry bath;
Reading your samples
Reading your samples
Prepare to read the absorbance of your samples in a microplate reader;
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 200 µL of the content of each microtube to it's corresponding well in the microplate;
Read the absorbance of the samples at 532 nm in a microplate reader;
Calculating data and determinig results
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);
CITATION
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 factors
or
MCF = _FC1+ FC2 + FC3 + FC4 + FC5_
5(number of factors)
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
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 (50 µg or 0.05 mg in this protocol).
nmol TBARS/mg protein = nmol TBARS on the sample / 0.05 mg of proteins
Citations
Adrieli Sachett, Matheus Gallas-Lopes, Radharani Benvenutti, Greicy M M Conterato, Ana Herrmann, Angelo Piato. How to prepare zebrafish brain tissue samples for biochemical assays
https://protocols.io/view/how-to-prepare-zebrafish-brain-tissue-samples-for-bjkdkks6Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti, Ana Herrmann, Angelo Piato. Optimized protein quantification protocol for zebrafish brain tissue (Bradford method)
https://protocols.io/view/optimized-protein-quantification-protocol-for-zebr-bjnfkmbnStep 2
Adrieli Sachett, Matheus Gallas-Lopes, Radharani Benvenutti, Greicy M M Conterato, Ana Herrmann, Angelo Piato. How to prepare zebrafish brain tissue samples for biochemical assays
https://protocols.io/view/how-to-prepare-zebrafish-brain-tissue-samples-for-bjkdkks6Step 2.3
Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti, Ana Herrmann, Angelo Piato. Optimized protein quantification protocol for zebrafish brain tissue (Bradford method)
https://protocols.io/view/optimized-protein-quantification-protocol-for-zebr-bjnfkmbnStep 4
Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani Benvenutti, Ana Herrmann, Angelo Piato. Optimized protein quantification protocol for zebrafish brain tissue (Bradford method)
https://protocols.io/view/optimized-protein-quantification-protocol-for-zebr-bjnfkmbn