Evaluating the Effect of Thymoquinone on Keap-1/Nrf-2 Signaling, Oxidative Stress, and Behavior in Rats

Summary

This protocol demonstrates how to assess the effects of thymoquinone on the Keap-1/Nrf-2 pathway, oxidative stress biomarkers, and behavioral outcomes in a rat model of subchronic deltamethrin exposure.

Abstract

Recent developments in pesticide exposure research have increased interest in antioxidant-based protective strategies. Thymoquinone (TQ), a plant-derived bioactive compound, has been reported to exert protective effects against various toxic agents. This study describes a protocol to evaluate the effects of TQ on oxidative stress parameters, the Keap-1/Nrf-2 signaling pathway, and behavioral outcomes in rats exposed to low-dose Deltamethrin (DTM). Here, 24 adult male Wistar Albino rats (250 ± 20 g) were randomly assigned to four groups (n = 6/group): control, TQ, DTM, and DTM+TQ. DTM (1.28 mg/kg) and TQ (10 mg/kg) were administered intragastrically for 30 days. Body weight was monitored throughout the study. Locomotor activity and anxiety-like behavior were assessed using the open field test on day 31, and depression-like behavior was evaluated using the forced swim test on day 32. At the end of the experimental period, plasma and brain tissues were collected for biochemical, histopathological, and molecular analyses. Oxidative stress markers, including malondialdehyde, total nitric oxide, glutathione, and sulfhydryl group levels, were measured in plasma and cerebral cortex samples. Kelch-like ECH-associated protein 1 (Keap-1) and Nuclear factor erythroid 2–related factor 2 (Nrf-2) expression levels were analyzed using Western blotting and immunohistochemistry. This protocol provides a comprehensive and reproducible approach for investigating pesticide-induced neurotoxicity and the modulatory effects of antioxidant compounds.

Introduction

Insecticides are widely used in agriculture and domestic environments to control pests; however, chronic exposure to low doses can result in environmental contamination and adverse health effects in both humans and animals. Such exposure, particularly through contaminated food and water, has been associated with an increased risk of neurological disorders and other systemic toxicities¹.

Pyrethroids have largely replaced organophosphorus insecticides due to stricter regulations on the latter and their relatively lower mammalian toxicity. DTM, a type II pyrethroid containing an α-cyano group, is among the most commonly used insecticides worldwide and is considered highly effective against insects². Despite this, experimental studies have demonstrated that DTM exposure induces oxidative stress and neurotoxicity in animal models^3,4.

DTM is a highly lipophilic compound that readily crosses the blood–brain barrier and disrupts neuronal function by prolonging the opening of voltage-gated sodium channels. This effect leads to repetitive neuronal firing, increased intracellular sodium influx, and enhanced generation of reactive oxygen species, ultimately resulting in oxidative damage, inflammation, and behavioral alterations, particularly in vulnerable brain regions such as the hippocampus and cerebral cortex¹. Given the growing concern regarding pesticide-induced neurotoxicity, increasing attention has been directed toward antioxidant-based protective strategies derived from natural sources. TQ, a major bioactive constituent of Nigella sativa essential oil, has been reported to possess potent antioxidant, anti-inflammatory, and neuroprotective properties⁵. Several studies have demonstrated its beneficial effects against chemically induced oxidative stress and neurodegenerative processes^5,6,7,8,9.

One of the principal mechanisms underlying the protective effects of TQ involves modulation of the Keap-1/Nrf-2 signaling pathway. This pathway plays a central role in maintaining cellular redox homeostasis by regulating the expression of antioxidant and detoxifying enzymes. Under oxidative stress conditions, Keap-1-mediated repression of Nrf-2 is relieved, allowing Nrf-2 to translocate to the nucleus and activate antioxidant response element–dependent gene expression⁹.

Because DTM exposure induces alterations at molecular, biochemical, histological, and behavioral levels, a single analytical approach is insufficient to capture the full extent of its neurotoxic effects. Therefore, this protocol integrates behavioral assessments, oxidative stress biomarker analyses, histopathological evaluation, and molecular investigation of the Keap-1/Nrf-2 pathway to provide a comprehensive and reproducible framework for studying pesticide-induced neurotoxicity and the modulatory effects of antioxidant compounds such as TQ.

Taken together, the evidence summarized above highlights the need for an integrated experimental approach to clarify the mechanisms underlying DTM-induced neurotoxicity and to evaluate potential protective interventions. By combining behavioral assessments with biochemical, histopathological, and molecular analyses of the Keap-1/Nrf-2 pathway, the present protocol enables a multidimensional evaluation of oxidative stress–related neuronal damage. The interpretation of the experimental findings within this mechanistic framework emphasizes how TQ modulates redox signaling, attenuates tissue injury, and improves behavioral outcomes in the context of subchronic DTM exposure.

Protocol

The Institutional Animal Experimentation Local Ethics Committee approved the experimental protocol under registration number 68429034/35. The total duration of the study was 32 days. Prior to the experiments, animals were acclimatized to standard laboratory conditions. All experimental procedures were conducted in strict accordance with institutional and international animal ethics guidelines. The experimental materials used in this protocol are listed in the Table of Materials.

NOTE: Chemical and biological wastes were disposed of in accordance with the institution’s laboratory safety and waste management guidelines. Acids, bases, and organic solvents were collected in separate, labeled containers and delivered to the institutional hazardous waste unit. Biological materials and contaminated consumables were collected in biohazard containers, sterilized, and transferred to the appropriate institutional facility for final disposal.

1. Experimental design

1. Obtain 24 adult male Wistar Albino rats weighing 250 ± 20 g from the Animal Experiment Laboratory. House each rat individually in cages at 24 ± 2 °C under a 12 h light/dark cycle, with ad libitum access to standard rat chow and tap water.
2. Randomly divide the animals into four groups (n = 6 per group): Group 1: Control (C), Group 2: TQ, Group 3: DTM, Group 4: DTM + TQ.
3. Dissolve DTM (98% purity) in corn oil and administer at a dose of 1.28 mg/kg via intragastric gavage for 30 days.
4. Dissolve TQ at a dose of 10 mg/kg in corn oil and administer via intragastric gavage for 30 days. Prepare all drug solutions fresh daily and administer at a total volume of 1 mL/kg.
5. In the combined treatment group, administer TQ immediately after DTM. Administer corn oil alone to the control group to account for gavage-related stress.
   NOTE: The DTM dose was selected based on previous literature to induce oxidative stress without causing morbidity and corresponds to approximately 1/100 of the LD₅₀¹.

2. Measurement of body weight

1. Measure body weight on days 1, 10, 20, and 30 of the study (Figure 1).

3. Behavioral parameters

1. Perform all behavioral tests under standardized conditions to ensure consistency. Record both the Forced Swim Test (FST) and Open Field Test (OFT) using an overhead camera providing a full view of the testing apparatus. Maintain illumination at approximately 100–150 lux. Define immobility as the absence of active movement, except for minimal movements required to maintain posture and respiration. Clean all testing arenas with 70% ethanol between animals to prevent olfactory influences.
2. FST
   1. Perform FST on day 32 to evaluate depression-like behavior. Conduct a 15-min pre-test session 24 h before the 5-min test session to habituate the animals to the water environment and minimize novelty-induced stress. Do not perform behavioral scoring during the pre-test session.
   2. Individually place rats into cylindrical containers (diameter: 35 cm, height: 50 cm) containing 30 cm of water maintained at 25 ± 1 °C for 5 min.
   3. Record swimming, climbing, and immobility durations using a video camera. Quantify behavioral parameters in seconds from video recordings¹⁰.
   4. Define behaviors as follows:
      Immobility: Consider the animal immobile when it remains floating without active movements, making only minimal movements necessary to keep its head above water.
      Swimming: Define swimming as active horizontal movement throughout the cylinder with coordinated movements of all four paws.
      Climbing: Define climbing as vigorous upward-directed movements of the forepaws against the cylinder wall while the animal remains in a vertical position.
   5. If the animal remains completely motionless, score the behavior as immobility according to the predefined criteria.
   6. Do not apply external stimulation or course correction during the test session. Allow the animal to behave freely for the entire 5-min period unless intervention is required for safety reasons (e.g., risk of drowning).
3. OFT
   1. Conduct OFT on day 31 to assess locomotor activity and anxiety-like behavior¹¹. Record individual movements for 5 min in an arena measuring 90 x 35 cm, divided into 24 equal units by black lines.
   2. Quantify the following behavioral parameters from video recordings by an investigator blinded to group allocation:
      The number of crossings with all four paws (locomotor activity)
      Time spent in the center and peripheral zones (anxiety-like behavior)
   3. If an animal remains completely motionless, record the behavior as immobility and assign zero crossings for that period.
   4. Do not apply external stimulation, repositioning, or course correction during the test session.
   5. Allow the animal to behave freely for the entire 5-min period unless intervention is required for safety reasons.
   6. Clean the arena with 70% ethanol between animals to eliminate olfactory cues.

4. Assessment of oxidative stress markers

1. Perform all procedures on day 32. Deeply anesthetize rats with intramuscular xylazine (5 mg/kg) and ketamine (45 mg/kg). Confirm deep anesthesia by the absence of the pedal withdrawal reflex. Collect approximately 3–5 mL of blood via cardiac puncture into EDTA-coated tubes.
2. Complete euthanasia by exsanguination under deep anesthesia and confirm death before tissue collection. Centrifuge blood samples at 3,000 x g for 10–15 min at 4 °C. Carefully collect plasma without disturbing the buffy coat and store aliquots at −80 °C. Dissect the brain rapidly on ice. Separate the left cerebral hemisphere and hippocampus for biochemical analyses and snap-freeze in liquid nitrogen (−80 °C storage). Fix the right cerebral hemisphere in 10% neutral buffered formalin for histological evaluation.
3. Plasma Oxidative Stress Analyses
   1. Plasma Lipid Peroxidation (TBARS Assay)
      1. Centrifuge plasma samples (3,000 x g, 10 min, 4 °C). Mix 0.5 mL plasma with 1 mL of TCA–TBA–HCl reagent (15% trichloroacetic acid (TCA), 0.375% thiobarbituric acid (TBA), 0.25 N Hydrochloric acid (HCl)). Centrifuge at 1,000 x g for 10 min and transfer the supernatant to glass tubes. Add 50 µL of 0.02% Butylated hydroxytoluene (BHT) to prevent artificial oxidation.
      2. Heat at 100 °C for 15 min, cool to room temperature, centrifuge at 1,000 x g for 10 min, and measure supernatant absorbance at 532 nm¹⁰. Calculate MDA concentration using Beer–Lambert law
         ε = 1.56 × 10⁵ M⁻¹ cm⁻¹, l = 1 cm and express as nmol/mL plasma.
   2. Plasma Thiol (RSH) Levels
      1. Mix 0.5 mL of plasma with Tris-HCl buffer (100 mM, pH 8.2) containing 1% Sodium dodecyl sulfate (SDS) and 2 mM Ethylenediaminetetraacetic acid (EDTA). Incubate 5 min at 25 °C and centrifuge (3,000 x g, 10 min, 4 °C). Use the supernatant for analysis.
      2. Add 0.3 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and incubate at 37 °C for 15 min. Measure absorbance at 412 nm¹⁰. Calculate RSH concentrations using ε = 13,600 M⁻¹ cm⁻¹ and express as µmol/L plasma.
   3. Plasma NOx Levels (Griess Reaction)
      1. Deproteinize plasma with 0.3 M Sodium hydroxide (NaOH) and 5% zinc sulfate (ZnSO₄). Centrifuge at 20,000 x g for 5 min and collect the supernatant.
      2. Prepare nitrate stock solutions (100, 10, and 1 mmol/L) and generate a standard curve (1–100 µmol/L). Load 100 µL of supernatant (duplicate wells) into a microplate.
      3. Add 100 µL of vanadium (III) chloride (VCl₃) followed by 50 µL of sulfanilamide and 50 µL of N-(1-naphthyl) ethylenediamine. Incubate 30–45 min at room temperature and measure absorbance at 540 nm¹⁰. Determine NOx concentrations from the standard curve and express as µmol/L plasma.
4. Tissue oxidative stress analyses
   1. Use approximately 100 mg of left cerebral hemisphere tissue for all biochemical analyses.
   2. Tissue lipid peroxidation (TBARS Assay)
      1. Homogenize tissue (1:10 w/v) in ice-cold 10% TCA. Add an equal volume of 0.67% TBA and heat at 100 °C for 15 min. Cool and centrifuge (3,000 x g, 10 min, 4 °C)¹⁰. Measure supernatant absorbance at 535 nm. Calculate MDA using ε = 1.56 × 10⁵ M⁻¹ cm⁻¹ and express as nmol/g wet tissue.
   3. Tissue glutathione (GSH) levels
      1. Homogenize tissue in 10% TCA (1:10 w/v) and centrifuge (3,000 x g, 10 min, 4 °C). Mix 0.5 mL of supernatant with 2 mL of DTNB solution (0.4 mg/mL in 1% sodium citrate)¹⁰. Measure absorbance at 412 nm immediately. Calculate GSH using ε = 13,600 M⁻¹ cm⁻¹ and express as µmol/g wet tissue.
   4. Tissue NOx levels (Griess Reaction)
      1. Homogenize tissue in phosphate-buffered saline (PBS) (1:5 w/v) and centrifuge (2,000 x g, 5 min). Deproteinize supernatant with 0.3 M NaOH and 5% ZnSO₄. Centrifuge (3,000 x g, 20 min) and collect the clear supernatant.
      2. Prepare nitrate standards (1–100 µmol/L). Load 100 µL of supernatant in duplicate wells. Add VCl₃ and Griess reagents as described above. Incubate 45 min at 37 °C and measure absorbance at 540 nm¹⁰. Determine concentrations from the standard curve and express as µmol/g tissue.
         CAUTION: Procedures involving hazardous chemicals, including TCA, HCl, NaOH, and organic solvents, were performed in a certified chemical fume hood. Appropriate personal protective equipment (PPE), such as laboratory coats, chemical-resistant gloves, protective eyewear, and, when necessary, face shields, was used throughout all experiments. All chemicals were handled according to institutional safety guidelines and relevant material safety data sheets (MSDS).

5. Determination of Keap-1 and Nrf-2 Levels by Western blot analysis

1. Cut hippocampal tissue into 50 mg pieces and place into homogenization tubes containing 500 µL of RIPA buffer supplemented with protease inhibitor cocktail.
2. Add an appropriate amount of magnetic beads and homogenize at 1,000 x g for two cycles. Centrifuge at 14,000 x g for 10 min at 4 °C.
3. Transfer the supernatant to clean microcentrifuge tubes and centrifuge again under the same conditions. Keep samples on ice and determine protein concentrations.
4. Measure protein concentrations using a fluorometer with the Protein BR Assay Kit, following the manufacturer’s instructions.
5. Prepare 10% SDS–PAGE gels for electrophoresis. Adjust protein concentration to 30 µg/µL.
6. Add 4x Laemmli buffer at a 3:1 ratio and supplement with 10% β-mercaptoethanol. Heat samples at 95 °C for 5 min, then briefly centrifuge.
7. Load 5 µL of protein marker and 30 µg protein per lane. Run gels at 110 V for 60–90 min.
8. Activate PVDF membranes in 100% methanol for 3–5 min, then equilibrate in 1× transfer buffer for 10 min at room temperature before transfer.
9. Wet filter papers and sponges with transfer buffer and assemble the transfer sandwich carefully, ensuring complete removal of air bubbles. Perform protein transfer using a wet transfer system at 100 V for 90 min at 4 °C (constant voltage).
10. Block membranes in 3% Bovine Serum Albumin (BSA) for 1 h at room temperature on an orbital shaker. Incubate membranes overnight at 4 °C with primary antibodies diluted in 3% BSA (Keap-1 and Nrf-2, 1:3,000; β-actin, 1:1,000).
11. Wash membranes 5x for 5 min with 1 × Tris-buffered saline with 0.1% Tween 20 detergent (TBS-T). Incubate with secondary antibody (1:15,000) for 1 h at room temperature in the dark.
12. Wash membranes as described and incubate with an enhanced chemiluminescence (ECL) substrate (e.g., luminol-based HRP substrate) according to the manufacturer’s instructions (mixing equal volumes of reagent A and B immediately before use). Apply sufficient substrate to completely cover the membrane surface (approximately 1 mL per membrane) and incubate for 1–2 min at room temperature.
13. Detect chemiluminescent signals using a digital imaging system equipped for chemiluminescence detection (no specific excitation wavelength required, as light emission results from HRP–luminol reaction). Acquire images using automatic exposure settings while ensuring non-saturated bands. Maintain identical exposure parameters for all samples within the same blot.
14. Perform densitometric analysis using Image analysis software under identical ROI selection, background subtraction, and normalization parameters. Normalize Keap-1 and Nrf-2 band intensities to the corresponding housekeeping protein β-actin. Export normalized values as .csv files for statistical analysis.

6. Histopathological analysis

1. Fix the right cerebral hemisphere in 10% neutral formalin for 72 h. Dehydrate through a graded ethanol series (70%–100%), embed in paraffin, and section at 5 µm.
   Obtain coronal brain sections at the hippocampal and entorhinal cortex levels according to the stereotaxic coordinates defined in Paxinos & Watson, The Rat Brain in Stereotaxic Coordinates (Academic Press). Use the interactive rat brain atlas viewer (https://labs.gaidi.ca/rat-brain-atlas to verify and refine anatomical localization¹¹.
2. Prepare four consecutive coronal sections (5 µm thickness) from paraffin-embedded brain tissue using standard histological procedures. Stain the sections with hematoxylin and eosin (H&E) according to conventional protocols¹². Examine all slides under a light microscope at 200× magnification. Ensure that the histological evaluation is performed by a blinded investigator to minimize observer bias¹³.
   NOTE: This procedure ensured consistent anatomical comparison across groups.
3. Semiquantitatively assess neuronal loss, cellular edema, nuclear pyknosis, inflammatory infiltration, and vascular congestion in 10 random fields per section.
4. Calculate mean scores for each parameter per group. Scoring criteria were as follows: 0 (none, <5%), 1 (mild, <33%), 2 (moderate, 33–66%), and 3 (severe, >66%; Table 1).

7. Immunohistochemical analysis of Nrf-2 and Keap-1 reactivity in the hippocampus and entorhinal cortex

1. Prepare 5 µm thick sections from the hippocampus and entorhinal cortex and mount them on glass slides.
2. Deparaffinize and rehydrate the sections, then wash in phosphate-buffered saline (PBS) at room temperature.
3. Incubate sections with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. Wash sections with PBS
4. Perform antigen retrieval by heating the slides in sodium citrate buffer (pH 6.0) for 10–15 min. Allow the slides to cool for 30 min, then wash with PBS.
5. Incubate sections again with 3% hydrogen peroxide for 15 min to ensure complete peroxidase inhibition. Wash sections with PBS.
6. Apply a blocking solution for 10 min to reduce nonspecific binding. Incubate the sections overnight at 4 °C with primary antibodies against Nrf-2 (1:100 dilution) and Keap-1 (1:200 dilution). Wash the sections with PBS after incubation.
7. Incubate sections with a suitable secondary antibody for 30 min at room temperature. Wash sections with PBS.
8. Apply streptavidin–peroxidase to the sections and incubate for 20 min at room temperature in a humidified chamber (maintain 95–100% relative humidity using a sealed incubation box containing moistened filter paper). Wash the sections with PBS following incubation.
9. Apply DAB chromogen and monitor color development under a light microscope at 100× magnification. Stop the reaction by rinsing the slides with tap water.
10. Counterstain the sections with hematoxylin diluted 1:9 for 5 min at room temperature. Rinse thoroughly with PBS and distilled water. Mount the sections using a permanent mounting medium.
11. Examine the stained sections under a light microscope. Capture photomicrographs at ×400 magnification from 16 representative fields per section.
12. Quantify Nrf-2 and Keap-1 immunoreactivity using image analysis software. Record and analyze the quantified data for further statistical evaluation.
13. For immunohistochemical analysis, open the images in ImageJ and calibrate using the Set Scale tool. Convert all images to 8-bit grayscale to standardize intensity measurements. Manually define regions of interest (ROIs) for positively stained areas and adjacent background regions and save them in the ROI Manager.
14. Quantify staining intensity using consistent H-DAB color deconvolution, followed by identical thresholding parameters across all samples. Measure mean gray value, optical density, and percentage of positive area.
15. For cell-based analysis, segment thresholded images using the Watershed tool, and quantify positive cells using Analyze Particles. All data were exported as .csv files for statistical analysis.

8. Statistical analysis

1. Perform statistical analyses using SPSS version 29.0. Enter raw data in the Data View tab, with each row representing an individual animal and variables defined in the Variable View tab; the experimental group was coded as a categorical variable (Control, TQ, DTM, and DTM+TQ).
2. Assess data normality using the Shapiro–Wilk test by selecting Analyze > Descriptive Statistics > Explore, assigning outcome variables to the Dependent List and Group to the Factor List, and enabling Normality plots with tests. For normally distributed variables, conduct group comparisons using one-way analysis of variance (ANOVA) via Analyze > Compare Means > One-Way ANOVA, with descriptive statistics and Levene’s test for homogeneity of variances enabled under Options.
3. When a significant main effect was detected, apply Tukey’s post hoc test through the Post Hoc menu to identify pairwise group differences. Use Levene’s test results to confirm variance homogeneity (p > 0.05).
4. For histopathological scoring data that did not meet parametric assumptions, perform nonparametric analysis using the Kruskal–Wallis test by selecting Analyze > Nonparametric Tests > Legacy Dialogs > K Independent Samples, followed by Dunn’s post hoc test with Bonferroni correction using the Independent Samples nonparametric test module.
5. Export statistical outputs and summary tables as .xls or .csv files, results were expressed as mean ± standard deviation (SD), and a p-value < 0.05 was considered statistically significant.

Results

Body weight measurements
Body weight was recorded on days 1, 10, 20, and 30. Rats in the C and TQ groups showed a progressive increase in body weight over the experimental period, with no significant difference between these groups on day 30. In contrast, rats exposed to DTM exhibited a significant reduction in body weight compared with controls. Co-administration of TQ attenuated DTM-associated weight loss. No mortality was observed during the study period (Figure 1).

Discussion

This study demonstrates that subchronic oral exposure to DTM induces consistent behavioral, biochemical, molecular, and histopathological alterations in rats and that TQ effectively mitigates these effects when administered concomitantly. By integrating standardized behavioral tests with oxidative stress profiling, pathway-level analysis of Keap-1/Nrf-2 signaling, and histopathological evaluation, the protocol provides a robust and reproducible framework for assessing pesticide-induced neurotoxicity and antioxidant-based...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5,5'-Dithiobis(2-nitrobenzoic acid)	Thermo Scientific	69-78-3
Benchtop centrifuge NF048	Nüve	43560
Beta_Actin	ThermoFisher Scientific, United States	7D2C10
BSA solution	Proteintech, United States)	66201-1-Ig
Butylated hydroxytoluene	Sigma Aldrich	128-37-0
Dijital Homojenizatör	Daihan Scientific	https://www.artlaboratuv arcihazlari.com/urun/dai han-hg-15d-dijital-homoje nizator-set-a?srsltid=Afm BOorx_az06ulcHpSzKZs uP8-47Za4BkvLOM22Xh p00tgwWhJ-86Vc
DTM	Bayer	TR01426058O
Hematoxylin–eosin	Bio Optica	W01030708
Hydrochloric acid	Merck	7647-01-0
Image J software program	NIH; Washington, U.S.A.	https://imagej.net/ij/
Immunohistochemical staining kit	Lab VisionTM UltraVisionTM Large Volume Detection System: anti-polyvalent	HRP, TA-125-HL
Leica Autocut 14051956472	Germany	https://www.leicabiosystems .com/histology-equipment/mi crotomes/histocore-autocut/
Light microscope	Nikon, Eclipse	Ni-U, 940728
N-(1-Naphthyl)ethylenediamine	Thermo Scientific	1465-25-4
Nano Microplate Reader	BMG LABTECH	SPECTROstar Nano
phosphate-buffered saline	Merck	P4417
Primary antibodies Keap-1	Proteintech	Cat No. 10503-2-AP, 1:200
Primary antibodies Nrf-2	Proteintech	Cat No. 16396-1-AP, 1/100
Qubit Protein BR Assay Kit	ThermoFisher Scientific-INVITROGEN	Q33211
sodium citrate	Merck	03-04-6132
Sodium Dodecyl Sulfate	Thermo Scientific	151-21-3
sodium hydroxide	Merck	1310-73-2
Sodium nitrate	Merck	7631-99-4
Streptavidin Peroxidase	Thermo Scientific	SHRP248-B
sulfanilamide	Thermo Scientific	63-74-1
thiobarbituric acid	Merck	504-17-6
TQ	CAYMAN	490-91-5
Tricarballylic acid, 99%	Thermo Scientific Chemicals	139360500
Trichloroacetic acid	Thermo Scientific Chemicals	76-03-9
Tris-HCl	Thermo Scientific	1185-53-1
Vanadium(III) chloride	Sigma Aldrich	7718-98-1
West Pico PLUS Chemiluminescent Substrate and the iBright 750	Thermo Fisher Scientific system	https://www.clinxsci.com/product /mini_chemiluminescence_imagi ng_system?gad_source=1&gad _campaignid=16224184202&gbr aid=0AAAAAoN6UOOtoB8UD0d eumBAMD8Xi-1l-&gclid=CjwKCAj wgeLHBhBuEiwAL5gNEXtK2CNS LS1GqllMuNgkrFYDaW7yK585njQ jFja485Fkwj9lRUEKDBoCgLoQA vD_BwE
Zinc sulfate solution	Merck	7733-02-0