Name: Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules (Video) | JoVE Journal
Uploaded: 2022-09-16T14:10:00.000+00:00
Duration: 7 min 1 s
Description: Explore zebrafish modeling of allergic reactions to tick saliva biomolecules in alpha-Gal syndrome research. This method enables study of immune mechanisms and protective antibody responses using Danio rerio.

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5 minutes

We would like to thank members of the SaBio group for their collaboration in the experimental design and technical assistance with the fish experimental facility and Juan Galcer&#225;n S&#225;ez (IN-CSIC-UMH, Spain) for providing zebrafish. This work was supported by Ministerio de Ciencia e Innovaci&#243;n/Agencia Estatal de Investigaci&#243;n MCIN/AEI/10.13039/501100011033, Spain and EU-FEDER (Grant BIOGAL PID2020-116761GB-I00). Marinela Contreras is funded by the Ministerio de Ciencia, Innovaci&#243;n y Universidades, Spain, grant IJC2020-042710-I.

All methods described here have been approved by the Ethics Committee on Animal Experimentation of the University of Castilla La Mancha under the study &#34;Evaluation of the immune response to inactivated M. bovis vaccine and challenge with M. marinum in the zebrafish model number PR-2017-05-12.&#34;
Ticks were obtained from the laboratory colony, where representative samples of ticks in the colony were tested by PCR for common tick pathogensto confirm the absence of pathogens, and maintained at the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences (IP BC CAS), Czech Republic.All animal experiments were performed in accordance with the Animal Protection Law of the Czech Republic No. 246/1992 Sb (ethics approval No. 34/2018).
1. Zebrafish treatment
NOTE: The trial is designed to evaluate allergic reactions and the immune response in zebrafish treated with tick saliva in response to mammalian meat consumption.
<ol>
	<li>Treat the fish (as explained in section 4) with tick saliva, commercial Gala1-3Gal-BSA 3 (&#945;-Gal) (see Table of Materials), used as a positive control, with phosphate-buffered saline (PBS) as a negative control. Adult zebrafish is randomly distributed into three gender-balanced groups (Figure 1). 
	NOTE: Any other desired compound related with AGS can be evaluated using this model.</li>
</ol>
2. &#8203; Ixodes ricinus tick saliva extraction
<ol>
	<li>Use semi-engorged pathogen-free female ticks fed for 6-7 days on guinea pigs.</li>
	<li>Treat the tick with 5 &#181;L of a 2% (wt/vol) solution of pilocarpine hydrochloride in PBS (see Table of Materials) at pH 7.4 into the hemocoel using a 50 &#181;L syringe with a 0.33 mm needle as previously was described28 to induce tick saliva production. 
	NOTE: Ticks are handled using forceps; be cautious to not apply too much strength when gripping them.</li>
	<li>Collect saliva using a 10 &#181;L tip mounted on a micropipette.
	<ol>
		<li>Introduce the tip inside the tick hypostome carefully.</li>
		<li>Keep the saliva in a 1.5 mL tube on ice, pool it, and store it at -80 &#176;C as previously described27.</li>
	</ol>
	</li>
	<li>Determine the saliva protein concentration, to establish the amount of protein to be injected into the fish as in previous studies27 using a BCA Protein Assay Kit (see Table of Materials) following the manufacturer&#39;s recommendations.</li>
</ol>
3. Maintenance of zebrafish
<ol>
	<li>Maintain zebrafish in a flow-through water system at 27 &#176;C with a light/dark cycle of 14 h/10 h (Figure 2).</li>
	<li>Feed the fish twice daily at 9:30 a.m. and 1:30 p.m. with dry fish feed (50-70 &#956;g/fish) until day 2.</li>
	<li>Feed the fish twice daily at 9:30 a.m. and 1:30 p.m. with dry dog feed (50-70 &#956;g/fish) from day 2 after the treatment injection until the end of the experiment</li>
</ol>
4. Zebrafish injection
<ol>
	<li>Select 10 fish per group with a similar ratio of females/males and similar weight. 
	NOTE: Group 1 contains fish injected with PBS, group 2 contains fish injected with tick saliva, and group 3 contains fish injected with &#945;-Gal.</li>
	<li>Anesthetize the fish briefly by immersion in 0.02% tricaine methanesulphonate (MS-222) (Movie 1). 
	NOTE: Properly anesthetized fish show normal breathing and no swimming, while they could be placed at the bottom of the water tank or floating. Each fish must be individually anesthetized to avoid possible physiological damage.</li>
	<li>Capture the anesthetized fish using a fishing net.</li>
	<li>Place the fish on its half side using forceps or hands carefully, on a wet sponge, with the caudal fin on the right side to inject the compounds in the same direction in order to control the lesions.</li>
	<li>Inject groups of fish intradermally, as in previous studies26, in the muscle at 5 mm to the caudal fin and at a 45&#176; angle in relation to the body of the fish (Movie 2). Use the appropriate treatment at days 0, 3, and 8 as previously described27&#160;with a 100 &#181;L syringe fitted with a 1 cm, 29 G needle with 1 &#181;L (with 9 &#181;g/&#181;L of protein) of I. ricinus saliva in 10 &#181;L of PBS (tick saliva), 5 &#181;g of &#945;-Gal in 10 &#181;L PBS (&#945;-Gal)27,&#160;and 10 &#181;L of PBS (Figure 3). 
	NOTE: Handling must be done quickly and carefully to avoid any physical damage to the animal. 
	Other biomolecules in tick saliva can be evaluated following this protocol.</li>
	<li>Place the treated fish back in a freshwater tank without anesthesia for recovery. 
	&#8203;NOTE: All the fish of the same group can be placed in the same water tank for recovery.</li>
</ol>
5. Zebrafish feeding
<ol>
	<li>Mash the dog food with a mortar and pestle.</li>
	<li>Feed 50-70 &#956;g/fish twice daily at 9:30 a.m. and 1:30 p.m. with dry fish feed until day 2.</li>
	<li>Feed 50-70 &#956;g/fish twice daily at 9:30 a.m. and 1:30 p.m. with mashed dog feed from day 2 after the treatment injection until the end of the experiment on day 8. 
	&#8203;NOTE: If immunity markers or antibody titers to &#945;-Gal or IgE antibody in response to the treatments or the feed throughout the different inoculations are to be evaluated, feeding would be necessary until the end of the experiment.</li>
</ol>
6. Evaluation of allergic reactions, lesions, and behavior in zebrafish
<ol>
	<li>Examine the hemorrhagic type of allergic reactions (skin redness, discoloration, and hemorrhage) using a magnifier or stereomicroscope for accuracy and indicate the location of their appearance on the fish following the categorization included in Table 1 (Figure 4A). 
	NOTE: The allergic reactions presented in Figure 4 appeared after the injection of tick saliva and the consumption of feed containing red meat. Therefore, the reactions described are the type of reactions associated with AGS, as similar reactions appear in the clinical context.
	<ol>
		<li>Observe if any reaction appears after treatments and while administering food twice a day while the fish are in the water tank.</li>
	</ol>
	</li>
	<li>Examine the fish behavior by evaluating the changes27 in swimming patterns (mobility, speed, standing motionless at the bottom of the water tank, and zigzag swimming) following the categorization included in Table 1.</li>
	<li>Evaluate accumulated mortality, reporting the number of dead fish including the time/day of death (Figure 4B). 
	NOTE: All parameters are evaluated right after treatment or after the change of feed and followed daily until the end of the experiment on day 8 categorizing qualitative variables (Table 1). As a recommendation, this evaluation should be conducted by a professional with knowledge on zebrafish to consider behavioral changes based on their background and experience working with this animal model.</li>
	<li>Calculate the number of zebrafish per day with reported allergic reactions, abnormal behavior, and feeding changes in each group and compare between groups by a one-way ANOVA test.</li>
</ol>
7. Sample collection
<ol>
	<li>Euthanize the fish by immersion in 0.04% MS-222 on day 8. 
	NOTE: Also collect the samples from the fish that die from allergic reactions during the trial.</li>
	<li>Fix the fish on a paraffin plate with pins.</li>
	<li>Collect serum from the gill-blood vessels29 of the fish immediately after euthanasia, when the gills are still irrigated with blood, using a 0.5 mL syringe fitted with a 1 cm, 29 G needle. Store it in a 1.5 mL tube at -20 &#176;C until use (Movie 3).</li>
	<li>Cut the fish sagittally with a scalpel blade and evaluate the internal lesions (hemorrhagic lesions or granulomas)27,30&#160;if they appear. 
	NOTE: Lesions do not necessarily appear but must be registered if they do.</li>
	<li>Collect the intestine (Movie 4) and kidney (Movie 5) from each fish in separate empty 1.5 mL tubes, as previously described31, and store them at -80 C (Figure 4C).</li>
	<li>Extract total RNA from the zebrafish intestine and kidney samples using an RNA purification kit (see Table of Materials).</li>
	<li>Analyze the expression of genes related to immune response as previously described30,32 (see Table 2 for primer sequences) in zebrafish, performing a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) using a reverse transcription mix for RT-qPCR (see Table of Materials), according to the manufacturer&#39;s instructions. Normalize the mRNA cT values against D. rerio GAPDH, and compare between groups (fish treated with saliva, &#945;-Gal, and the PBS-treated groups)using a Student t-test with unequal variance.</li>
	<li>Determine IgM antibody titers that recognize &#945;-Gal in zebrafish in serum samples by ELISA as described previously27,30. Record the antibody titers as O.D.450 nm values, using a plate reader, and compare between groups (fish treated with saliva, &#945;-Gal, and the PBS-treated groups)using a Student t-test with unequal variance. 
	NOTE: Determination of IgM antibody titers and expression gene analysis is optional and conducted only if immunological information is required. RT-qPCR mix is a first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR.</li>
</ol>

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<li>Lu, M. -W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interferon+response+is+involved+in+nervous+necrosis+virus+acute+and+persistent+infection+in+zebrafish+infection+model%5BTitle%5D)+AND+%22Molecular+Immunology%22%5BJournal%5D)">The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model.</a> Molecular Immunology. 45 (4), 1146-1152 (2008).</li>
<li>Saralahti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+zebrafish+model+for+pneumococcal+pathogenesis%5BTitle%5D)+AND+%22Developmental+and+Comparative+Immunology%22%5BJournal%5D)">Adult zebrafish model for pneumococcal pathogenesis.</a> Developmental and Comparative Immunology. 42 (2), 345-353 (2014).</li>
<li>Gore, A. V., Pillay, L. M., Venero Galanternik, M., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish%3A+A+fintastic+model+for+hematopoietic+development+and+disease%5BTitle%5D)+AND+%22Wiley+Interdisciplinary+Reviews.+Developmental+Biology%22%5BJournal%5D)">The zebrafish: A fintastic model for hematopoietic development and disease.</a> Wiley Interdisciplinary Reviews. Developmental Biology. 7 (3), 312(2018).</li>
<li>Katoch, S., Patial, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish%3A+An+emerging+model+system+to+study+liver+diseases+and+related+drug+discovery%5BTitle%5D)+AND+%22Journal+of+Applied+Toxicology%22%5BJournal%5D)">Zebrafish: An emerging model system to study liver diseases and related drug discovery.</a> Journal of Applied Toxicology. 41 (1), 33-51 (2021).</li>
<li>Kalueff, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+a+comprehensive+catalog+of+zebrafish+behavior+1.0+and+beyond%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.</a> Zebrafish. 10 (1), 70-86 (2013).</li>
<li>Xin, N., Jiang, Y., Liu, S., Zhou, Y., Cheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+prednisolone+on+behavior+and+hypothalamic-pituitary-interrenal+axis+activity+in+zebrafish%5BTitle%5D)+AND+%22Environmental+Toxicology+and+Pharmacology%22%5BJournal%5D)">Effects of prednisolone on behavior and hypothalamic-pituitary-interrenal axis activity in zebrafish.</a> Environmental Toxicology and Pharmacology. 75, 103325(2020).</li>
<li>Aleström, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish%3A+Housing+and+husbandry+recommendations%5BTitle%5D)+AND+%22Laboratory+Animals%22%5BJournal%5D)">Zebrafish: Housing and husbandry recommendations.</a> Laboratory Animals. 54 (3), 213-224 (2020).</li>
</ol>

The authors have nothing to disclose.

Zebrafish is a cost-effective and easy-to-handle model that also has been a very feasible tool for the study of molecular mechanisms of the immune response, pathogen diseases, novel drug testing, and vaccination and protection against infections33,34,35. The study on the behavior of zebrafish is useful since previous studies have found that some fish species remain motionless at the bottom of the tank when they are stressed, whi...

Ticks are vectors of pathogens that cause diseases and are also the cause of allergic reactions, affecting the health of humans and animals worldwide1,2. During tick feeding, biomolecules in tick saliva, especially proteins and lipids, facilitate the feeding of these ectoparasites, avoiding host defenses3. Some saliva biomolecules with glycan Gal&#945;1-3Gal&#946;1-(3)4GlcNAc-R (&#945;-Gal) modifications lead to the production of high anti-&#945;-Gal IgE antibody levels after the tick bite, only in some individuals, which is known as &#945;-Gal Syndrome (AGS)4. This is a disease associated with IgE-mediated allergy that may result in anaphylaxis to tick bites, non-primate mammalian meat consumption, and some drugs such as cetuximab5. Reactions to &#945;-Gal are often severe and sometimes could be fatal6,7,8,9,10,11,12,13,14,15.
The &#945;-Gal is found in all mammals except for Old World monkeys, apes, and humans that do not have the ability to synthesize &#945;-Gal13. However, pathogens such as bacteria and protozoa express this glycan on their surface, which can induce the production of high amounts of anti-&#945;-Gal IgM/IgG antibodies and may be a protective mechanism against these pathogens16,17. However, the production of anti-&#945;-Gal antibodies increases the risk of developing IgE-mediated anti-&#945;-Gal allergies7,13. Natural anti-&#945;-Gal antibodies produced in humans, mainly of the IgM/IgG subtypes, could be associated with this modification present in bacteria from the gut microbiota16. AGS can be a challenging clinical diagnosis, as the main diagnostic method at the moment is based on a clinical history of delayed allergic reactions, especially associated with food allergies (i.e., pruritus, localized hives, or recurrent angioedema to anaphylaxis, urticaria, and gastrointestinal symptoms) and the measurement of IgE anti-&#945;-Gal antibody levels9. Current findings suggest tick bites constitute one of the principal risks in the appearance of AGS18,19, a 20-fold or greater increase in IgE levels to &#945;-Gal following a tick bite19, a history of tick bites in patients with AGS20,21,22, the existence of antibodies reactive to tick antigens in AGS patients19, and that anti-&#945;-Gal IgE are strongly related to anti-tick IgE levels19,23 but further studies are needed to assess which biomolecules are actually involved.
In addition, another possible scenario is patients who present strong allergic reactions to tick bites and high levels of anti-&#945;-Gal IgE antibodies but are tolerant to mammalian meat consumption12. Therefore, mammalian meat allergy could be a particular type of tick bite-related allergy. The principal tick species associated with AGS include Amblyomma americanum (USA), Amblyomma sculptum (Brazil), Amblyomma testudinarium and Haemaphysalis longicornis (Japan), Ixodes holocyclus (Australia), and Ixodes ricinus (the main vector of Lyme borreliosis in Europe)11,24.
The only model that has been used to evaluate IgE production related with tick bites is the mouse model genetically modified with the gene for &#945;&#8722;1,3-galactosyltransferase knocked out (&#945;-Gal KO) mice25,26 because like other mammals, mice also express &#945;-Gal on proteins and lipids and do not produce IgE to &#945;-Gal. However, zebrafish (Danio rerio) is a useful model for biomedical research applied to mammals because it shares many anatomical similarities with mammals and, like humans, is also unable to synthesize &#945;-Gal. Since &#945;-Gal is not produced naturally in zebrafish, it is an affordable model, easy to manipulate, and allows a high sample size for the study of &#945;-Gal-related allergic reactions.
In this study, zebrafish is used as a model organism to characterize and describe local allergic reactions, behavioral patterns, and the molecular mechanisms associated with response to percutaneous sensitization to tick saliva26,27 and subsequent mammalian meat consumption. For this purpose, fish are exposed to tick saliva by intradermal injection and then are fed with dog feed, that contains mammalian meat-derived products suitable for animal use which contains &#945;-Gal27, then possible related allergic reactions are evaluated. This method may be applied to the study of other biomolecules related to allergic processes, especially those related to AGS.

<table><tbody><tr><td>1.5 mL tube</td><td>VWR</td><td>525-0990</td><td></td></tr><tr><td>All Prep DNA/RNA</td><td>Qiagen</td><td>80284</td><td></td></tr><tr><td>Aquatics facilities</td><td></td><td></td><td></td></tr><tr><td>BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>Disection set</td><td>VWR</td><td>631-1279</td><td></td></tr><tr><td>Dog Food - Red Classic</td><td>Acana</td><td></td><td></td></tr><tr><td>ELISA plates-96 well</td><td>Thermo Fisher Scientific</td><td>10547781</td><td></td></tr><tr><td>Gala1-3Gal-BSA 3 (&amp;alpha;-Gal)&amp;nbsp;</td><td>Dextra</td><td>NGP0203</td><td></td></tr><tr><td>iScript Reverse Transcription Supermix</td><td>Supermix</td><td>1708840</td><td></td></tr><tr><td>Microliter syringes</td><td>Hamilton</td><td>7638-01</td><td></td></tr><tr><td>Plate reader</td><td>any</td><td></td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-50TAB</td><td></td></tr><tr><td>pilocarpine hydrochloride&amp;nbsp;</td><td>Sigma</td><td>P6503</td><td></td></tr><tr><td>Pipette tip P10&amp;nbsp;</td><td>VWR</td><td>613-0364</td><td></td></tr><tr><td>Pipette tip P1000</td><td>VWR</td><td>613-0359</td><td></td></tr><tr><td>Premium food tropical fish</td><td>DAPC</td><td></td><td></td></tr><tr><td>Sponge Animal Holder&amp;nbsp;</td><td></td><td></td><td>Made from scrap foam</td></tr><tr><td>Stereomicroscope</td><td>any</td><td></td><td></td></tr><tr><td>Thermal Cycler Real-Time PCR</td><td>any</td><td></td><td></td></tr><tr><td>Tricaine methanesulphonate (MS-222)</td><td>Sigma</td><td>E10521</td><td></td></tr></tbody></table>

zebrafish animal model for the study of allergic reactions in response to tick saliva biomolecules

Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules

Ticks are arthropod vectors that cause disease by pathogen transmission and whose bites could be related to allergic reactions impacting human health worldwide. In some individuals, high levels of immunoglobulin E antibodies against the glycan Gal&#945;1-3Gal&#946;1-(3)4GlcNAc-R (&#945;-Gal) have been induced by tick bites. Anaphylactic reactions mediated by glycoproteins and glycolipids containing the glycan &#945;-Gal, present in tick saliva, are related to alpha-Gal syndrome (AGS) or mammalian meat allergy. Zebrafish (Danio rerio) has become a widely used vertebrate model for the study of different pathologies. In this study, zebrafish was used as a model for the study of allergic reactions in response to &#945;-Gal and mammalian meat consumption because, like humans, they do not synthesize this glycan. For this purpose, behavioral patterns and hemorrhagic anaphylactic-type allergic reactions in response to Ixodes ricinus tick saliva and mammalian meat consumption was evaluated. This experimental approach allows the obtention of valid data that support the zebrafish animal model for the study of tick-borne allergies including AGS.

The protocol presented here is based on several aspects of previously published experiments27,30 and results performed in our laboratory where the zebrafish model is established and validated for the study of AGS and the immune response to α-Gal because both humans and zebrafish do not synthesize this molecule13. This model allows the characterization and evaluation of a variety of allergic reactions as a result of the host response to tic...

Here, zebrafish (Danio rerio) is used as a model to study allergic reactions and immune responses related to alpha-Gal syndrome (AGS) by evaluating allergic reactions to tick saliva and mammalian meat consumption.

Ticks are arthropod vectors that cause disease by pathogen transmission and whose bites could be related to allergic reactions impacting human health ...

zebrafish-animal-model-for-study-allergic-reactions-response-to-tick

Research

JoVE Journal

Immunology and Infection

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease.  In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs.  We also demonstrate saliva collection from female I. scapularis adults.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host's immune responses with qPCR.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This article presents the methodology for exposing humans to larval Ixodes scapularis for clinical research. The technique is relatively simple, tolerable by the research volunteers, and can be modified according to experimental needs. Such research involving human subjects must be conducted under clinical study protocols approved by the appropriate regulatory authorities.

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

Here, we present a protocol to collect saliva, haemolymph, salivary glands, and midgut, following the dissection of an individual fed tick, to study tissue-specific localization of tick-borne pathogens to advance understanding of vector competence and pathogen-microbiome interactions.

Extraction of Saliva, Haemolymph, Salivary Glands, and Midgut from Individual Ticks (Acari: Ixodidae)

This method outlines the development of an in vitro tick blood-feeding system incorporating a 3D-printed chamber and a laboratory-fabricated silicone membrane. The system enables artificial feeding of ticks, allowing for easy sampling at various feeding stages and utilizing low volumes of blood, providing an invaluable tool for studying ixodid ticks.

Tick Blood Feeding through an Artificial Membrane on a 3D-Printed Chamber in Ixodid Tick Research

To model how inflammatory signals influence neurodevelopment in genetically susceptible organisms, we present a protocol for microinjecting IL-6 into the duct of Cuvier (common cardinal vein) of zebrafish larvae, enabling controlled induction of inflammation and assessment of its developmental impact in the context of tuberous sclerosis complex (TSC).

Microinjection of Interleukin-6 into the Bloodstream of TSC Zebrafish Larvae to Study Neuropsychiatric Disorder-Like Behaviors

Explore zebrafish modeling of allergic reactions to tick saliva biomolecules in alpha-Gal syndrome research. This method enables study of immune mechanisms and protective antibody responses using Danio rerio.

Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules

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