Whole-Mount Immunostaining of Tyrosine Hydroxylase for Dopaminergic Neuron Analysis in Zebrafish Larvae

Materials and reagents

Biological materials

1. 96 h post-fertilization (hpf) AB wild-type zebrafish larvae

Reagents

1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)

2. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911)

3. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S7907)

4. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P0662)

5. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465)

6. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148)

7. Methanol (MeOH) (Sigma-Aldrich, catalog number: 34860)

8. Ethanol (EtOH) (Sigma-Aldrich, catalog number: E7023)

9. Acetone (Sigma-Aldrich, catalog number: 179124)

10. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

11. Tween-20 (MP Biomedicals, catalog number: TWEEN201)

12. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473)

13. Hydrogen peroxide (H₂O₂) (Sigma-Aldrich, catalog number: 216763)

14. Bovine serum albumin (BSA) (Fisher Scientific, catalog number: BP9702)

15. Tyrosine hydroxylase antibody (Merck, catalog number: MAB318)

16. Alexa Fluor 488 secondary conjugated antibody (Jackson ImmunoResearch Europe Ltd., catalog number: 115-545-003)

17. Glycerol (Sigma-Aldrich, catalog number: 356352-M)

18. Sodium carbonate (Na₂CO₃) (Sigma-Aldrich, catalog number: S7795)

19. Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S6297)

20. MilliQ water

21. Xylol (Sigma-Aldrich, catalog number: 1.08298)

Solutions

1. 10× phosphate-buffered saline (PBS) (see Recipes)

2. 1 N NaOH (see Recipes)

3. 4% PFA (see Recipes)

4. Ethanol (EtOH)/xylol solution (1:1 v/v) (see Recipes)

5. 80% acetone (see Recipes)

6. PTwx (see Recipes)

7. 100 mM KOH

8. 3% H₂O₂ (see Recipes)

9. Bleaching solution (see Recipes)

10. AbDil solution (see Recipes)

11. Glycerol solution (see Recipes)

12. 0.5 M carbonate buffer (pH 9.0) (see Recipes)

13. Buffered glycerol (see Recipes)

Recipes

1. 10× PBS

Note: Adjust to neutral pH by adding acid or base as appropriate until the solution is pH 7.2–7.4. Sterilize by autoclaving for 20 min and store at 4 °C. Dilute in MilliQ water to 1× (v/v) before use.

2. 1 N NaOH

3. 4% PFA

Note: Heat the solution to approximately 60 °C under stirring to facilitate dissolution. Avoid overheating and do not exceed 60 °C, as higher temperatures may lead to paraformaldehyde degradation. Slowly add 1 N NaOH until the solution clears. After, adjust pH to around 7.4 with diluted HCl and adjust the volume of the solution to 100 mL.

4. EtOH/xylol solution (1:1 v/v)

5. 80% acetone

6. PTwx

7. 100 mM KOH

8. 3% H₂O₂

9. Bleaching solution

10. AbDil solution

11. Glycerol solution

12. 0.5 M carbonate buffer (pH 9)

13. Buffered glycerol

Laboratory supplies

1. 1.5 mL microcentrifuge tubes

2. 2.0 mL microcentrifuge tubes

3. Routine glassware and consumables (e.g., pipette tips, conical tubes, etc.)

4. Glass microcapillaries (Merck, catalog number: P2049)

Equipment

1. Rotator (e.g., Labnet Mini LabRoller Rotator)

2. Variable volume micropipettes

3. Stir plate

4. pH meter

5. Stereomicroscope (e.g., Olympus, model: SZX7)

6. Fluorescence microscope (e.g., Olympus, model: IX51 inverted fluorescence microscope)

Software and datasets

1. Image analysis software (e.g., ImageJ [21])

2. Statistical software (e.g., GraphPad Prism [22])

Procedure

A. Zebrafish larvae collection

1. AB wild-type zebrafish embryos are generated by pairwise mating using conventional methods [23].

2. Disinfect embryos [24] and sort them for viability at 2–3 h post-fertilization on a stereomicroscope.

3. Expose for 96 h to a selected drug/neurotoxin in E3 medium [25], under laboratory conditions [26], and according to standard guidelines [27].

B. Larvae fixation

1. At the end of the exposure period, collect 10 larvae in 1.5 mL centrifuge tubes and add 500 μL of 4% PFA.

2. Maintain at 4 °C for 14–18 h.

Caution: Fixation of zebrafish larvae in 4% PFA can be achieved at room temperature (RT) for 2–4 h. However, optimal tissue morphology has been achieved by overnight fixation at 4 °C. Over-fixation may lead to a higher background.

Caution: Fixed samples can be stored in 4% PFA at 4 °C for short periods (up to 24–48 h). Prolonged storage in PFA may lead to over-fixation and increased background fluorescence. Therefore, for longer storage, samples should be transferred to 100% methanol and kept at -20 °C.

Caution: Fixatives are broadly classified into aldehyde-based fixatives—such as formaldehyde, glutaraldehyde, and formalin—that work by creating bonds between lysine residues to cross-link sample proteins, and alcohol-based organic solvents—such as methanol, ethanol, and acetone—that work by dehydrating the sample material, which causes protein denaturation and precipitation. These latter fixatives do not require a separate permeabilization procedure. However, alcohol-based fixation can wash away soluble proteins and change the tertiary structure of target epitopes due to hydrophobic bond breaking. For this reason, aldehyde-based fixatives are preferred.

3. Wash three times in PBS for 5 min each at RT.

4. Store in 500 μL of 100% MeOH at -20 °C for at least 6 h.

Critical: Methanol is used to delipidate larvae. The larvae may be stored for several months and probably longer in this solution.

Pause point: Stop and continue the next day.

C. Sample permeabilization and bleaching

1. Replace MeOH by washing twice in 1 mL of EtOH.

2. Incubate samples in 1 mL of EtOH/xylol (1:1 v/v) for 1 h under rocking conditions.

Caution: Xylol is used as a rapid clearer for specimens that have been previously dehydrated in alcohol. However, as a relatively hazardous solvent, another xylene-free method can be adapted [28].

3. Wash twice in absolute ethanol for 5 min.

4. Rehydrate the sample for 15 min in 1 mL of descending ethanol concentrations (90%, 75%, 50%, 25%, and then H₂O) containing 0.1% Tween 20.

5. Wash twice in H₂O for 5 min.

6. Permeabilize the samples in 1 mL of 80% acetone at -20 °C for 40 min without shaking [29].

Critical: Acetone must be pre-chilled at -20 °C.

Caution: Acetone permeates the larvae, allowing the antibody to pass through the skin. Other permeabilizing reagents, such as Proteinase K or trypsin, can be used, and the amount used depends on the antibody of interest. However, acetone has been shown to be more effective than other permeabilization agents [20]. To aid in permeabilization, the skin of the larvae can be removed (by peeling it back with forceps).

7. Wash larvae twice in PTwx for 5 min.

8. Bleach larvae in bleaching solution for approximately 30 min or until pigmentation has completely disappeared.

Critical: Bleaching can damage tissue, so care must be taken to minimize the time larvae spend in the bleaching solution.

9. Wash larvae twice in PTwx for 5 min.

Pause point: If the protocol cannot be immediately continued, samples can be left in this solution for at least one week if stored refrigerated.

D. Blocking and antibody incubation

1. Block in AbDil solution under rocking conditions. Alternatively, blocking can be achieved by 2-h incubation at RT or overnight at 4 °C.

2. Rinse twice in PTwx.

3. Incubate in primary antibody (1:500) in AbDil overnight at 4 °C under rocking conditions.

Critical: The volume of antibody to be used should always be sufficient to cover the larvae, which should be between 100 and 500 μL per condition.

Critical: To achieve adequate immunostaining, shorter incubation times at 37 °C, longer incubation times (1–2 h) at room temperature, or extended overnight incubation at 4 °C can be used.

Caution: Antibodies can be reused. However, do not reuse more than twice. If reusing, store the removed primary antibody solution in an appropriate container at 4 °C.

4. Wash off excessive primary antibody 6 times for 5 min in PTwx.

5. Incubate in secondary antibody (1:500) in AbDil overnight at 4 °C on a shaker.

Critical: Secondary antibodies are photosensitive. Keep them protected from light as much as possible.

6. Wash three times in PTwx for 5 min each.

7. Rinse in PBS and store in 50% glycerol/PBS at 4 °C until imaging.

E. Imaging and analysis

1. Replace 50% glycerol/PBS with 70% glycerol/PBS and allow the larvae to sink to the bottom of the tube.

Critical: Before moving on to the next glycerol concentration, allow larvae to sink to the bottom of the tube, which can take up to 1 h.

Critical: Glycerol raises the refractive index of the mounting media, so brighter and higher resolution images will be obtained in a higher percentage of glycerol.

Pause point: Samples can be stored in 70% glycerol for up to one week at 4 °C. It is, however, strongly advised to image samples as soon as possible.

2. Replace 70% glycerol/PBS with buffered glycerol and store in the dark to preserve fluorescent signal.

Note: Although other antifading agents could be used with higher antifading properties [30,31], glycerol is a common reagent in the laboratory and is easy to obtain.

3. Aspirate the larvae into a microcapillary.

Note: The use of a microcapillary facilitates precise handling and positioning of the larvae, allowing easy orientation of the specimen under the microscope and improving consistency during imaging.

4. Image the samples using a fluorescence microscope with appropriate filter settings for the fluorophore used (e.g., FITC/Alexa Fluor 488).

Critical: Before imaging experimental groups, first acquire images from control samples to define optimal microscope settings (e.g., exposure time, gain, and illumination). Once established, these settings should be kept constant for all subsequent samples to ensure comparability between conditions.

5. Acquire images focusing on the brain regions containing dopaminergic neurons.

6. After imaging, use ImageJ to analyze the data. Open ImageJ and draw a region of interest (ROI) in the brain of zebrafish larvae (Figure 1). Go to Analyse > Set Measurements to configure the desired parameters and check if Area and Mean Grey Value are selected. Go to Analyse > Measure, and a pop-up window will appear with your measurements. Copy the value under Mean, which corresponds to the mean fluorescence of your ROI, to a spreadsheet. Move the ROI to the background, repeat the measurement, and correct the fluorescence obtained.

Figure 1. Main steps in conducting fluorescence analysis from the whole-mount assay in zebrafish larvae. (A) Fluorescence analysis using the ImageJ software. A representative image of zebrafish larvae with fluorescent signals is shown. In addition, an ImageJ window displaying the ROI and the fluorescence intensity is shown. Dopaminergic neurons in zebrafish larvae are primarily organized in clusters within the preoptic area (PO), posterior tuberculum (PT), and hypothalamus (H) [32]. (B) Quantification of tyrosine hydroxylase (TH) in zebrafish larvae exposed to a neurotoxin, 6-OHDA. Data is expressed as mean and standard deviation of five independent replicates, normalized to the control values. Asterisks (**) indicate significant differences when compared to the control (Ctrl) group (t-test, p < 0.01).

Data analysis

Fluorescence images were analyzed using ImageJ software. For each zebrafish larva, a region of interest (ROI) was manually defined in the brain area containing dopaminergic neurons. Mean fluorescence intensity was measured for each ROI after background subtraction by placing an equivalent ROI in an adjacent nonfluorescent area. For each experimental condition, five independent biological replicates (each containing 10 individual larvae) were analyzed. One ROI per larva was quantified, and the average of the 10 animals was measured as n = 1. Larvae presenting tissue damage, poor antibody penetration, or imaging artifacts were excluded from the analysis. No additional outlier removal was performed. Fluorescence intensity values were normalized to the control group and expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism version 9.1 (GraphPad Software, USA). Comparisons between treated and control groups were conducted using Student’s t-test. Differences were considered statistically significant when p < 0.05. No advanced computational or programming skills are required to perform the data analysis.

Validation of protocol

This protocol (or parts of it) has been used and validated in the following research article(s):

• Gomes et al. [36]. Protective effects of 24-epibrassinolide against the 6-OHDA zebrafish model of Parkinson's disease. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology (Figure 3 and Figure S1). https://doi.org/10.1016/j.cbpc.2023.109630

• Carneiro et al. [37]. Amelioration of 6-OHDA-induced Parkinson’s symptoms in zebrafish larvae by an almond skin acetonic extract. International Journal of Molecular Sciences (Figure 2). https://doi.org/10.3390/ijms27062590

General notes and troubleshooting

This protocol provides a robust and reproducible approach for whole-mount immunostaining of zebrafish larvae, with particular suitability for the detection of dopaminergic neurons via tyrosine hydroxylase (TH) analysis. However, several factors may influence the outcome and should be considered. For instance, the antibody quality and specificity, which may vary between batches and suppliers, can result in different outcomes. Optimization of antibody concentration and incubation conditions may be required when adapting the protocol to different targets. In addition, the efficiency of antibody penetration is highly dependent on proper permeabilization, as insufficient permeabilization may result in weak or uneven staining, while excessive treatment can damage tissue integrity.

Pigmentation in zebrafish larvae can also interfere with signal detection. Although the bleaching step reduces this issue, prolonged exposure may compromise tissue morphology. Therefore, careful monitoring of bleaching duration is recommended. Notably, this protocol does not require the use of phenylthiourea (PTU) to inhibit pigmentation, thereby avoiding potential developmental effects associated with PTU treatment [33–35].

In addition, this protocol is optimized for early larval stages, and its direct application to later developmental stages or adult tissues may require further adjustments, particularly in permeabilization and clearing steps. Furthermore, variability in imaging conditions, including microscope settings and sample orientation, may affect fluorescence quantification. To ensure consistency, it is recommended to standardize imaging parameters and maintain consistent positioning of larvae across samples.

Despite these considerations, the protocol can be adapted to detect other neuronal or non-neuronal markers, provided that appropriate optimization of antibody conditions is performed.

Acknowledgments

This work is supported by National Funds by FCT – Portuguese Foundation for Science and Technology, under the projects UID/04033/2025: Centre for the Research and Technology of Agro-Environmental and Biological Sciences (https://doi.org/10.54499/UID/04033/2025) and LA/P/0126/2020 (https://doi.org/10.54499/LA/P/0126/2020) and 2021.00458.CEECIND.

This protocol was used in [36,37].

Competing interests

The author declares no conflicts of interest.

Ethical considerations

All experiments were conducted prior to the embryos reaching the free-feeding stage; therefore, ethical approval was not required under the EU Animal Protection Directive 2010/63/EU.

References

1. Coons, A. H., Creech, H. J. and Jones, R. N. (1941). Immunological Properties of an Antibody Containing a Fluorescent Group. Exp Biol Med. 47(2): 200–202. https://doi.org/10.3181/00379727-47-13084p
2. Duraiyan, J., Govindarajan, R., Kaliyappan, K. and Palanisamy, M. (2012). Applications of immunohistochemistry. J Pharm Bioallied Sci. 4(Suppl 2): S307–309. https://doi.org/10.4103/0975-7406.100281
3. Matos, L. L., Trufelli, D. C., de Matos, M. G. and da Silva Pinhal, M. A. (2010). Immunohistochemistry as an important tool in biomarkers detection and clinical practice. Biomark Insights. 5: 9–20. https://doi.org/10.4137/bmi.s2185
4. Grigorev, I. and Korzhevskii, D. (2018). Current Technologies for Fixation of Biological Material for Immunohistochemical Analysis (Review). Sovremennye Tehnologii V Medicine. 10(2): 156–164. https://doi.org/10.17691/stm2018.10.2.19
5. Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T. and Ueda, H. R. (2016). Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling. Annu Rev Cell Dev Biol. 32(1): 713–741. https://doi.org/10.1146/annurev-cellbio-111315-125001
6. Piña, R., Santos-Díaz, A. I., Orta-Salazar, E., Aguilar-Vazquez, A. R., Mantellero, C. A., Acosta-Galeana, I., Estrada-Mondragon, A., Prior-Gonzalez, M., Martinez-Cruz, J. I., Rosas-Arellano, A., et al. (2022). Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence. Int J Mol Sci. 23(3): 1426. https://doi.org/10.3390/ijms23031426
7. Im, K., Mareninov, S., Diaz, M. F. P. and Yong, W. H. (2019). An Introduction to Performing Immunofluorescence Staining. Methods Mol Biol.: 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26
8. Bauer, B., Mally, A. and Liedtke, D. (2021). Zebrafish Embryos and Larvae as Alternative Animal Models for Toxicity Testing. Int J Mol Sci. 22(24): 13417. https://doi.org/10.3390/ijms222413417
9. Choi, T. Y., Choi, T. I., Lee, Y. R., Choe, S. K. and Kim, C. H. (2021). Zebrafish as an animal model for biomedical research. Exp Mol Med. 53(3): 310–317. https://doi.org/10.1038/s12276-021-00571-5
10. Santos, D., Monteiro, S. M. and Luzio, A. (2018). General Whole-Mount Immunohistochemistry of Zebrafish (Danio rerio) Embryos and Larvae Protocol. Methods Mol Biol. 1797: 365–371. https://doi.org/10.1007/978-1-4939-7883-0_19
11. Hammond-Weinberger, D. R. and ZeRuth, G. T. (2020). Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae. J Visualized Exp.: e3791/60575. https://doi.org/10.3791/60575
12. Montero, C. (2003). The Antigen-Antibody Reaction in Immunohistochemistry. J Histochem Cytochem. 51(1): 1–4. https://doi.org/10.1177/002215540305100101
13. Ramos-Vara, J. A. and Miller, M. A. (2013). When Tissue Antigens and Antibodies Get Along. Vet Pathol. 51(1): 42–87. https://doi.org/10.1177/0300985813505879
14. Monné Rodríguez, J. M., Frisk, A. L., Kreutzer, R., Lemarchand, T., Lezmi, S., Saravanan, C., Stierstorfer, B., Thuilliez, C., Vezzali, E., Wieczorek, G., et al. (2023). European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development. Toxicol Pathol. 51(3): 92–111. https://doi.org/10.1177/01926233231178282
15. Silvestri, L., Costantini, I., Sacconi, L. and Pavone, F. S. (2016). Clearing of fixed tissue: a review from a microscopist’s perspective. J Biomed Opt. 21(8): 081205. https://doi.org/10.1117/1.jbo.21.8.081205
16. Ueda, H. R., Ertürk, A., Chung, K., Gradinaru, V., Chédotal, A., Tomancak, P. and Keller, P. J. (2020). Tissue clearing and its applications in neuroscience. Nat Rev Neurosci. 21(2): 61–79. https://doi.org/10.1038/s41583-019-0250-1
17. Mathias, J. R., Saxena, M. T. and Mumm, J. S. (2012). Advances In Zebrafish Chemical Screening Technologies. Future Med Chem. 4(14): 1811–1822. https://doi.org/10.4155/fmc.12.115
18. Choe, C. P., Choi, S. Y., Kee, Y., Kim, M. J., Kim, S. H., Lee, Y., Park, H. C. and Ro, H. (2021). Transgenic fluorescent zebrafish lines that have revolutionized biomedical research. Lab Anim Res. 37(1): 26. https://doi.org/10.1186/s42826-021-00103-2
19. Tolleson, C. and Claassen, D. (2012). The Function of Tyrosine Hydroxylase in the Normal and Parkinsonian Brain. CNS Neurol Disord Drug Targets. 11(4): 381–386. https://doi.org/10.2174/187152712800792794
20. Vauti, F., Stegemann, L. A., Vögele, V. and Köster, R. W. (2020). All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish. PLoS One. 15(8): e0237167. https://doi.org/10.1371/journal.pone.0237167
21. Abràmoff, M. D., Magalhães, P.J. and Ram, S.J. (2004). Image processing with ImageJ. Biophoton Int. 11(7): 36–42.
22. Swift, M. L. (1997). GraphPad Prism, Data Analysis, and Scientific Graphing. J Chem Inf Comput Sci. 37(2): 411–412. https://doi.org/10.1021/ci960402j
23. Nasiadka, A. and Clark, M. D. (2012). Zebrafish Breeding in the Laboratory Environment. ILAR J. 53(2): 161–168. https://doi.org/10.1093/ilar.53.2.161
24. Chang, C. T., Colicino, E. G., DiPaola, E. J., Al-Hasnawi, H. J. and Whipps, C. M. (2015). Evaluating the effectiveness of common disinfectants at preventing the propagation of Mycobacterium spp. isolated from zebrafish. Comp Biochem Physiol C Toxicol Pharmacol. 178: 45–50. https://doi.org/10.1016/j.cbpc.2015.09.008
25. Meyers, J. R. (2018). Zebrafish: Development of a Vertebrate Model Organism. Curr Protoc Essent Lab Tech. 16(1): e19. https://doi.org/10.1002/cpet.19
26. Aleström, P., D’Angelo, L., Midtlyng, P. J., Schorderet, D. F., Schulte-Merker, S., Sohm, F. and Warner, S. (2019). Zebrafish: Housing and husbandry recommendations. Lab Anim. 54(3): 213–224. https://doi.org/10.1177/0023677219869037
27. Busquet, F., Strecker, R., Rawlings, J. M., Belanger, S. E., Braunbeck, T., Carr, G. J., Cenijn, P., Fochtman, P., Gourmelon, A., Hübler, N., et al. (2014). OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing. Regul Toxicol Pharm. 69(3): 496–511. https://doi.org/10.1016/j.yrtph.2014.05.018
28. Metgud, R., Astekar, M., Soni, A., Naik, S. and Vanishree, M. (2013). Conventional xylene and xylene-free methods for routine histopathological preparation of tissue sections. Biotech Histochem. 88(5): 235–241. https://doi.org/10.3109/10520295.2013.764015
29. Oralová, V., Rosa, J. T., Soenens, M., Bek, J. W., Willaert, A., Witten, P. E. and Huysseune, A. (2019). Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafish. Biol Open. 8(5): e042374. https://doi.org/10.1242/bio.042374
30. Longin, A., Souchier, C., Ffrench, M. and Bryon, P. A. (1993). Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study. J Histochem Cytochem. 41(12): 1833–1840. https://doi.org/10.1177/41.12.8245431
31. Florijn, R. J., Slats, J., Tanke, H. J. and Raap, A. K. (1995). Analysis of antifading reagents for fluorescence microscopy. Cytometry. 19(2): 177–182. https://doi.org/10.1002/cyto.990190213
32. Schweitzer, J. and Driever, W. (2009). Development of the Dopamine Systems in Zebrafish. In: Development and Engineering of Dopamine Neurons. Pasterkamp, R.J., Smidt, M.P. and Burbach, J.P.H. (Eds.). Springer New York: New York, NY. 1–14. https://doi.org/10.1007/978-1-4419-0322-8_1
33. Bohnsack, B. L., Gallina, D. and Kahana, A. (2011). Phenothiourea Sensitizes Zebrafish Cranial Neural Crest and Extraocular Muscle Development to Changes in Retinoic Acid and IGF Signaling. PLoS One. 6(8): e22991. https://doi.org/10.1371/journal.pone.0022991
34. Elsalini, O. A. and Rohr, K. B. (2003). Phenylthiourea disrupts thyroid function in developing zebrafish. Dev Genes Evol. 212(12): 593–598. https://doi.org/10.1007/s00427-002-0279-3
35. Chen, X. K., Kwan, J. K., Chang, R. C. and Ma, A. H. (2020). 1-phenyl 2-thiourea (PTU) activates autophagy in zebrafish embryos. Autophagy. 17(5): 1222–1231. https://doi.org/10.1080/15548627.2020.1755119
36. Gomes, A., Monteiro, S. M., Venâncio, C. and Félix, L. (2023). Protective effects of 24-epibrassinolide against the 6-OHDA zebrafish model of Parkinson's disease. Comp Biochem Physiol C Toxicol Pharmacol. 269: 109630. https://doi.org/10.1016/j.cbpc.2023.109630
37. Carneiro, P., Pais, P., Oliveira, I. V., Monteiro, S. M., Venâncio, C. and Félix, L. (2026). Amelioration of 6-OHDA-Induced Parkinson’s Symptoms in Zebrafish Larvae by an Almond Skin Acetonic Extract. Int J Mol Sci. 27(6): 2590. https://doi.org/10.3390/ijms27062590