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Article de méthode

Imagerie et quantification des cellules immunitaires dans le microenvironnement tumoral à l'aide d'un modèle de neuroblastome chez le poisson-zèbre

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DOI :

10.3791/72028

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18 septembre 2026

 ,  ,  ,  ,  , 
1Departments of Pharmacology and Medicine, Section of Hematology and Medical Oncology, The Cancer Research Center, Boston University School of Medicine, 2Tulane University School of Medicine

* These authors contributed equally

Dans cet article

Résumé

Ce protocole décrit des méthodes pour générer des zebrafish transgéniques composés, imager les cellules immunitaires associées aux tumeurs chez des zebrafish larvaires et adultes, et quantifier la fréquence des cellules immunitaires au sein du microenvironnement tumoral.

Résumé

Le recrutement de cellules immunitaires vers le microenvironnement tumoral (MET) constitue un élément important de la régulation immunitaire et de la progression tumorale. L'étude des relations spatiales et temporelles entre les cellules immunitaires et les cellules tumorales in vivo peut fournir des informations sur les interactions au sein du MET. Toutefois, la visualisation et la quantification de ces interactions dans des organismes intacts restent difficiles à l’aide des approches classiques d’imagerie et d’histologie. Le poisson-zèbre constitue un modèle précieux pour la recherche sur le cancer en raison de sa facilité de manipulation génétique, de son développement rapide et de sa transparence optique au cours des stades précoces de développement, ce qui permet la visualisation de cellules marquées par fluorescence in vivo. Ici, nous présentons un protocole permettant de générer, d'identifier, d'imaginer et de quantifier des cellules immunitaires et tumorales marquées différemment par des marqueurs fluorescents dans un modèle de zebrafish du neuroblastome. Ce protocole comprend l'élevage et le criblage de zebrafish transgéniques doubles, l'imagerie en direct de larves, la préparation et l'imagerie de poissons adultes porteurs de tumeurs, ainsi que la quantification des cellules immunitaires entourant et infiltrant les régions tumorales. À l'aide de lignées rapporteuses transgéniques, CD4+ cellules immunitaires et MYCNdes cellules de neuroblastome peuvent être visualisées et analysées à différents stades de développement. Le flux de travail permet d'évaluer la localisation des cellules immunitaires par rapport aux tumeurs chez les poissons larvaires et adultes, et peut être utilisé pour comparer l'abondance des cellules immunitaires au cours des différents stades de la maladie. Ce protocole fournit une méthode reproductible pour l'imagerie et la quantification des cellules immunitaires au sein du microenvironnement tumoral (TME) in vivo en utilisant le poisson zèbre et pourrait faciliter l'étude des interactions entre le système immunitaire et la tumeur dans d'autres modèles de cancer.

Introduction

Le microenvironnement tumoral (TME) est un écosystème composé de cellules tumorales, immunitaires, de fibroblastes et vasculaires1. Le TME joue un rôle essentiel dans la progression du cancer, la réponse thérapeutique et l'immunité anti-tumorale. Au sein du TME, les cellules immunitaires sont des régulateurs clés du destin tumoral, participant soit aux réponses anti-tumorales, soit à l'induction d'un état d'immunosuppression2. Comprendre le recrutement, la distribution et la fonction des cellules immunitaires dans le TME est essentiel pour élucider les mécanismes par lesquels les tumeurs échappent à la surveillance immunitaire. Toutefois, les approches classiques d'étude du TME, telles que l'histologie en point final, la cytométrie en flux et le séquençage à la cellule unique, ne fournissent que des instantanés statiques et nécessitent souvent la destruction de la structure tissulaire, entraînant une perte d'informations spatiales et d'interactions entre cellules3,4,5,6,7. De plus, ces méthodes sont limitées dans leur capacité à visualiser et à quantifier les cellules immunitaires au sein du TME, en particulier durant les premiers stades du développement tumoral.

Les zebrafish (Danio rerio) constituent un modèle puissant pour la recherche sur le cancer en raison de leur faible coût, de leur développement rapide, de leur transparence relative et de leur forte conservation génétique8,9. Ces avantages offrent un système expérimental reproductible et évolutif qui permet des tailles d'échantillon importantes et un criblage microscopique efficace en un court laps de temps10. De manière importante, les zebrafish partagent une conservation immunologique significative avec les humains, incluant des composants clés des systèmes immunitaires inné et adaptatif, ce qui rend les découvertes pertinentes pour les maladies humaines11. Ces propriétés sont particulièrement utiles pour l'étude des cancers pédiatriques tels que le neuroblastome8. Par conséquent, de nombreuses lignées rapporteuses de l'immunité et modèles de cancer chez le zebrafish ont été développées11. Ainsi, le zebrafish constitue un modèle in vivo précieux pour étudier les interactions entre cellules tumorales et cellules immunitaires, offrant un aperçu de la manière dont la régulation immunitaire façonne le développement tumoral, notamment durant les stades précoces.

Les études antérieures sur le microenvironnement tumoral (TME) chez le poisson-zèbre se sont largement appuyées sur des modèles de xénogreffes tumorales combinés à une imagerie en point final pour évaluer la localisation des cellules immunitaires au sein des tumeurs12,13,14,15,16,17. Bien que ces méthodes fournissent des informations utiles, elles sont limitées par leur dépendance à l’imagerie bidimensionnelle, qui ne permet pas de rendre pleinement compte de l’organisation tridimensionnelle du TME ni des relations spatiales entre les cellules tumorales et les cellules immunitaires au cours du développement14. De plus, les modèles de xénogreffe tumorale impliquent la transplantation artificielle de cellules tumorales humaines et peuvent ne pas reproduire fidèlement l’initiation naturelle de la tumeur18. Ces modèles sont également généralement à court terme et ne permettent pas de saisir complètement la progression tumorale ni les modifications des relations spatiales entre cellules tumorales et cellules immunitaires au cours du développement14,19. En outre, cette approche offre une représentation limitée de l’organisation spatiale, du contexte développemental et de la pertinence physiologique du comportement des cellules immunitaires au sein du TME. Récemment, nous et d'autres chercheurs avons utilisé des poissons-zèbres transgéniques composés, dans lesquels les cellules tumorales et les cellules immunitaires sont marquées différemment par des marqueurs fluorescents, afin de visualiser les cellules immunitaires associées aux tumeurs20,21. Ces poissons transgéniques composés permettent d’étudier le TME tout au long du développement de l’organisme et de la tumeur, facilitant ainsi l’analyse des interactions entre le système immunitaire et la tumeur in vivo.

Ici, nous présentons une méthode qui utilise la microscopie confocale pour étudier les cellules immunitaires au sein du microenvironnement tumoral (TME) d'un modèle de neuroblastome chez le poisson zèbre. Dans cette approche, nous avons utilisé des lignées rapporteuses de poisson zèbre permettant de marquer les cellules immunitaires, ainsi qu'une lignée de poisson zèbre génétiquement modifiée surexprimant l'oncogène humain MYCN sous le promoteur de la dopamine-β-hydroxylase (dβh), ce qui conduit au développement d'un neuroblastome similaire à la maladie humaine20. Dans ce modèle transgénique double, les cellules tumorales et les cellules immunitaires sont marquées de manière différentielle par EGFP et mCherry, respectivement. Le promoteur dβh est actif dans les cellules de la lignée sympatho-surrénale et a été utilisé dans plusieurs modèles de neuroblastome chez le poisson zèbre22,23,24,25. De plus, nous avons utilisé un promoteur Cd4 dirigeant la fluorescence de mCherry afin de visualiser les cellules CD4+15. Une imagerie confocale avec acquisition en Z-stack a été appliquée pour visualiser le TME en trois dimensions à différents stades du développement tumoral, permettant d'évaluer les relations spatiales entre les cellules tumorales et immunitaires au sein d'un tissu intact. Cette méthode permet d'analyser l'organisation des cellules immunitaires par rapport aux cellules tumorales durant les différentes phases du développement tumoral.

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Protocole

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University Chobanian & Avedisian School of Medicine under protocol PROTO202100001. The overall workflow for generation of compound transgenic zebrafish, larval imaging, adult tissue preparation, and image analysis is summarized in Figure 1.

Zebrafish transgenic process diagram; fluorescence microscopy, embryo imaging, cryosection analysis.
Figure 1. Workflow for visualization and quantification of immune cells within the zebrafish tumor microenvironment (TME). Schematic overview of the experimental workflow. Immune-reporter zebrafish were crossed with tumor-prone zebrafish to generate compound transgenic offspring expressing fluorescent markers in both immune cells (red) and tumor cells (green). Embryos were screened by fluorescence microscopy to identify compound transgenic fish. Selected fish were either subjected to live confocal imaging during larval development (<21 days post-fertilization [dpf]) or processed by fixation, cryosectioning, and confocal imaging for fish older than 21 dpf. This workflow enables visualization and quantification of immune cells associated with the TME across multiple developmental stages. Part of the illustration was created using BioRender. Please click here to view a larger version of this figure.

1. Generation of Compound Transgenic Zebrafish Lines Through Breeding

  1. Select adult immune-reporter fish and tumor fish that are at least 3 months old for natural breeding.
    NOTE: For the representative results, Tg(cd4-1:mCherry) heterozygous fish were crossed with Tg(dβh:MYCN; dβh:EGFP) heterozygous fish to differentially label immune cells and tumor cells15,23.
  2. On the evening before breeding, fill a two-chamber mating tank two-thirds full with fish water.
  3. Place a grated top insert into the mating chamber. Use dividers to separate one male from one or two females.
  4. Leave the dividers in place overnight. Add artificial plants or other enrichment items to simulate natural breeding conditions.
    NOTE: A 1:2 male-to-female ratio increases the likelihood of successful spawning when breeding tumor male fish with immune-reporter fish. Do not breed two males with one female, as competition between males may occur.
  5. On the following morning, remove the dividers after the facility lights turn on.
  6. Tilt the grated top chamber slightly using the divider as a wedge to create a shallow water area. Allow the fish to spawn for at least 1 h.
  7. Collect eggs from the bottom of the mating chamber by pouring the water through a fine-mesh net.
  8. Transfer the eggs to a sterile Petri dish containing fish water at a density no greater than 1 egg per 50 mm2 (approximately 200 eggs per 100 mm dish).
  9. Remove dead and unfertilized eggs.
    NOTE: Healthy fertilized eggs are translucent and round. Remove cloudy, white, unfertilized, or deformed eggs.
  10. Double-check the fish to avoid mixing sexes or cross-contaminating transgenic tanks.
  11. Return the males and females to their respective tanks.
  12. Place the embryos in a 28.5°C incubator overnight. Do not exceed the embryo density specified in Step 1.8.
  13. Bleach the embryos on the following morning to remove contaminants.
    1. Prepare a working bleach solution consisting of 100 µL bleach in 200 mL system water. Prepare a neutralization solution consisting of 0.5 g sodium thiosulfate in 150–200 mL system water.
    2. Sort and transfer viable embryos into a mesh-net well insert. Submerge the insert in the bleach solution for 5 min.
    3. Transfer the insert directly into the sodium thiosulfate solution. Incubate for 2 min to neutralize residual bleach.
    4. Rinse the embryos in a final bath of fresh system water. Use a squirt bottle to gently transfer the embryos into clean petri dishes containing fresh system water.
  14. Return the embryos to the incubator after bleaching.
  15. Transfer larvae at 5–7 days post-fertilization (dpf) or later to a larger tank containing 5 ppt rotifer water for rearing.
    1. Prepare 5 ppt rotifer water by mixing 1 part stock rotifer water (15 ppt) with 2 parts zebrafish system water.
    2. Verify that the final salinity is 5 ppt using a conductivity meter before transferring the larvae.
      NOTE: Sorting EGFP+ MYCN larvae at 3–5 dpf facilitates identification of tumor-prone fish before rearing. Sorting MYCN embryos at this stage avoids false-negative identification because EGFP expression in neural crest cells may decrease later in development due to MYCN-induced apoptosis.

2. Screening Zebrafish for Fluorescent Markers

  1. Prepare a 0.4% (w/v) tricaine stock solution using system water.
  2. Transfer the fish to a clean petri dish containing only enough system water to cover the bottom.
  3. Add the tricaine stock solution to achieve a final concentration of approximately 0.005% (50 mg/L) for fish younger than 14 dpf or 0.012% (120 mg/L) for fish older than 14 dpf. Confirm adequate anesthesia by observing little to no voluntary movement.
  4. Remove excess water from the dish once the fish are anesthetized and no longer capable of swimming while maintaining visible opercular or cardiac movement.
  5. Screen the fish for fluorescence. Identify green or red fluorescence in the expected region (e.g., the interrenal gland region in MYCN fish).
  6. Separate the fish into groups using a stiff-bristled sorting tool.
    NOTE: Sort tumor-prone fish as early as possible because MYCN-induced apoptosis during later developmental stages may result in false-negative identification. Sort immune-reporter fish immediately before imaging at the larval stage or between 2 and 4 months of age for adult studies. This approach minimizes handling-associated stress and maximizes survival.
  7. Transfer positive and negative fish to separate tanks containing system water for continued rearing.
    NOTE: Step 3 describes handling and imaging of larvae younger than 21 dpf. Steps 4–6 describe fixation, tissue preparation, imaging, and image analysis of older fish.

3. Larval Live-Mounting and Imaging

  1. Place sorted larvae in a 100 mm petri dish containing tricaine fish water (see Step 2.3 for age-appropriate concentrations).
  2. Transfer the sedated larvae to a glass-bottom imaging dish.
  3. Use only enough solution to cover the larva and maintain sedation.
  4. Prepare 1%–1.5% low-melting agarose in fish water. Heat the agarose solution until fully dissolved.
  5. Allow the agarose to cool for 1–2 min. Maintain the agarose at 30°C using a heat block to prevent solidification prior to mounting.
  6. Position each larva laterally in the imaging dish.
  7. Apply approximately 30–200 µL of agarose over the larva to immobilize it. Use larger volumes for larger larvae and smaller volumes for smaller larvae.
  8. Once the agarose has solidified, add approximately 100 µL of 0.005% tricaine solution over the agarose.
  9. Image the larvae using a confocal microscope.
    1. Use a standard laser-scanning confocal microscope with a resonant scanner. Use a 40× objective with a numerical aperture greater than 1.10.
    2. Set the pinhole to 1.0 Airy unit (AU) for all imaging channels.
    3. Use a 488 nm laser for EGFP excitation and a 594 nm laser for mCherry excitation. Keep laser output below 25 mW to minimize photobleaching and phototoxicity. Adjust detector gain and laser power using a range indicator lookup table (LUT) to avoid pixel saturation and black clipping. Do not exceed a detector gain of 800 V because higher voltages introduce excessive noise.
    4. Use a voxel size of 0.42 × 0.42 × 6.0 µm (x, y, z) at a resolution of 1024 × 1024 pixels and a Z-step interval of 6.0 µm. Acquire optical sections continuously through the full volume of interest, typically spanning 90–120 µm in total imaging depth. Use highly sensitive photodetectors operating in 16-bit acquisition mode.
    5. Acquire images using unidirectional line scanning at a resonant scan frequency of 33.3 kHz (approximately 1.58 µs pixel dwell time). Use 2× or 4× averaging to reduce motion artifacts while limiting laser exposure.
      NOTE: Optimize tricaine exposure to maintain larval survival during imaging. After imaging, add fish water until the imaging dish is approximately half full.
  10. Add fresh fish water to the imaging dish immediately after imaging.
  11. Gently remove the larva from the agarose using a stiff-bristled stick and transfer the larva to a dish containing clean fish water for recovery.
  12. Return the fish to its original tank once normal swimming behavior resumes.
    NOTE: For fish older than 21 dpf, the skin overlying the tumor must be removed by cryosectioning to enable high-resolution confocal imaging.

4. Fish Fixation and Cryosectioning

  1. Euthanize fish in accordance with approved institutional animal care guidelines.
  2. Make a ventral incision extending from the anal pore to the pectoral girdle to expose the abdominal cavity and facilitate fixative penetration into internal tissues.
  3. Transfer the fish using forceps into a 1.5 mL tube containing 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) or a volume of fixative at least 10-fold greater than the tissue volume.
    NOTE: Avoid handling the region containing the tumor to preserve tissue integrity.
  4. Place the samples on a nutating rocker set to gentle continuous agitation (30 rpm) and incubate overnight for at least 12 h at 4°C.
    CAUTION: PFA is hazardous, toxic, and potentially carcinogenic. Wear appropriate personal protective equipment, including gloves and eye protection. Perform all procedures involving PFA in a fume hood and avoid inhalation of vapors.
  5. Remove the PFA and wash the samples at least four times with 1.5 mL of 1× PBS. Gently invert the tube two to three times during each wash.
  6. Transfer the fish to 30% sucrose prepared in PBS.
  7. Incubate the fish in a cold room overnight until the specimen sinks to the bottom of an upright tube (approximately 12 h), indicating adequate sucrose penetration.
  8. Remove excess sucrose solution by gently blotting the fish on a laboratory tissue.
  9. Transfer the fish to storage tubes.
  10. Store the samples at −80°C until imaging.
    NOTE: Process samples within 3 months to minimize fluorescent protein degradation and signal loss.
  11. Transport the fixed fish to the cryostat room on dry ice.
  12. Place the fish flat in a plastic mold with one sagittal side facing downward.
  13. Add Optimal Cutting Temperature (OCT) compound until the fish is fully covered.
  14. Freeze the mold at −20°C or in the cryostat until the OCT turns white.
    NOTE: Position the fish as flat as possible before freezing to facilitate consistent sectioning.
  15. Cryosection the zebrafish in the sagittal plane to remove the skin for subsequent whole-mount imaging.
    1. Cool the cryostat chuck.
    2. Apply room-temperature OCT to the chuck.
    3. Press the OCT fish block firmly into the OCT layer.
    4. Freeze the chuck and block until the newly applied OCT turns white.
    5. Mount the chuck in the cryostat.
    6. Cryosection the block at a thickness of 14 µm until the blade reaches the specimen.
      NOTE: Specimen shifting may occur during freezing, causing the fish to sit higher in the block than expected. Entry into the specimen is indicated by the appearance of translucent tissue replacing the opaque OCT background.
    7. Reduce the section thickness to 7 µm.
    8. Continue sectioning for one additional cut.
      NOTE: This procedure removes the skin while preserving a uniform sagittal profile across the specimen.
    9. Confirm the endpoint by identifying skin in the collected sections.
      ​NOTE: Skin appearance in sections provides a more reliable indicator than eye morphology. Perform this procedure only when the skin obscures imaging of the tumor microenvironment.
  16. Warm the chuck gently with your hands.
  17. Remove the OCT block from the chuck using forceps. Avoid disturbing the cut surface of the specimen.
  18. Trim excess OCT surrounding the fish while the OCT remains frozen.
  19. Remove the fish from the OCT block using forceps once the OCT softens to a viscous consistency.

5. Mounting and Imaging of Fish with Skin Removed

  1. Place the fish in a glass-bottom dish with the sectioned sagittal side facing the glass surface.
    NOTE: Maintain the orientation of the fish during handling. Identifying the sectioned side may become difficult after the fish is removed from the OCT block.
  2. Prepare 1%–1.5% low-melting agarose.
  3. Apply approximately 30–50 µL of agarose to the dorsal and ventral sides of the fish to secure the sample.
    NOTE: Avoid placing agarose beneath the fish because it can reduce image quality. Use only the minimum amount of agarose required for stabilization.
  4. Keep the sample on ice during transport. Image the sample within 2 h of mounting.
  5. Transport the sample to the confocal microscopy facility.
  6. Image the sample using confocal microscopy.
    1. Use a standard laser-scanning confocal microscope with a resonant scanner. A two-photon microscope may be used when autofluorescence limits image quality. Use a corrected 20× objective lens with a numerical aperture of at least 0.80.
    2. Set the pinhole to 1.0 Airy unit (AU) for all imaging channels.
    3. Use a 488 nm laser for EGFP excitation and a 594 nm laser for mCherry excitation. Keep laser output below 25 mW. Adjust detector gain and laser power using a range indicator lookup table (LUT) to avoid pixel saturation and black clipping. Do not exceed a detector gain of 800 V because higher voltages introduce excessive noise.
    4. Configure image acquisition parameters as follows.
      1. Use a voxel size of 0.42 × 0.42 × 6.0 µm (x, y, z) at a resolution of 1024 × 1024 pixels and a Z-step interval of 6.0 µm.
      2. Acquire spectral image stacks through the full volume of interest, typically spanning a total Z-stack acquisition range of 90–120 µm (15–20 optical sections).
      3. Initiate image acquisition in the fluid space immediately above the tissue surface and continue through the full target imaging depth to ensure complete coverage of the specimen.
      4. Acquire images using highly sensitive photodetectors operating in 16-bit acquisition mode.
    5. Acquire images using unidirectional line scanning at a resonant scan frequency of 33.3 kHz (approximately 1.58 µs pixel dwell time) with 8× line averaging.

6. Image Analysis and Quantification

  1. Image visualization using Fiji (version 1.54p).
    1. Open the confocal z-stack images.
    2. Merge colors to see channel overlay by selecting Image > Color > Merge Channels.
    3. Generate a projection of the full z-range containing the imaging region by selecting Image > Stacks > Z Project. Enter the slice range of interest, as well as Max Intensity projection type. This will display the 2D projection of the image.
      NOTE: For larval fish, it is feasible to capture the whole TME by confocal microscopy. Therefore, we recommend the volumetric normalization and quantification method for the TME analysis. For older fish, it is impossible to capture the whole TME by confocal microscopy; therefore, we recommend applying the area-based normalization and quantification method. Based on our analysis and quantification of the larval fish, both methods can provide reliable biological results.
  2. Volumetric normalization and quantification of the TME in the larval fish.
    1. Quantifying tumor volume using MATLAB R2022b (download the package from the Matlab website) and Bio-Formats package (download from the openmicroscopy website).
    2. The user runs a script that performs the following tasks:
      1. Prompt the user to select a .CZI file from their computer.
      2. Read the image and extract the data from the channel containing the tumor region.
      3. Identify the region for each slice that is the tumor region through a threshold of fluorescence (can be through user input or machine learning), keeping the same threshold for all analyzed images for consistency.
      4. Outline the tumor region and display the image with the tumor region outlined in a collage of all slices for user verification.
      5. Calculate the area of the tumor region per slice in pixels and convert to mm2 using the scale of the image and imaging settings.
      6. Multiply the area of the tumor region by the depth (stored in the file’s metadata) to get the volume of each slice.
      7. Sum the calculated values of each slice to obtain the total volume of the imaged tumor and its surrounding region in voxels or mm3.
      8. Scale both volume measurements by multiplying by 104 for tumor volume and 106 for the surrounding region. This will create a more reasonable cell number:volume ratio during normalization.
    3. Quantifying cell number.
      1. Open the confocal z-stack images.
      2. Merge colors to see channel overlay by selecting Image > Color > Merge Channels.
      3. Generate a 3D view of the full z-range containing the imaging region by selecting Image > Stacks > 3D Project. Select Brightest Point as the projection method and other settings to the desired values.
      4. Due to the small number of cells, manually count and tabulate the cells, separating cells located within the tumor boundary from those located outside the tumor boundary.
        NOTE: Cells on the boundary of the tumor region are counted as being outside.
    4. Normalization.
      1. For infiltrating cells inside the tumor region, divide the number of cells by the scaled tumor volume. For the cells outside the tumor region, divide the number of cells outside the tumor volume by the scaled volume of the full image minus the tumor volume.
      2. Generate a table that includes the following headings: “fish identification”, “tumor volume”, “# of cells inside tumor region”, “# of cells outside tumor region”, “normalized cells inside”, “normalized cells outside”.
  3. Area-based normalization and quantification of the TME.
    1. Define the tumor region of interest (ROI) using the Rectangle or Freehand selection tool to outline the tumor.
    2. Add the ROI to the ROI Manager by pressing T or by selecting Analyze > Tools > ROI Manager > Add.
    3. Measure the ROI area using Analyze > Measure.
    4. Confirm that Area is selected under Set Measurements before recording measurements.
    5. Save the ROI in the ROI Manager.
    6. Apply the same ROI to all images using ROI Manager > More > Multi-Measure.
    7. Do not resize the ROI between images.
    8. Manually count cells within the ROI. If cells are more, count cells using the Cell Counter plugin by selecting Plugins > Analyze > Cell Counter.
    9. Mark each counted cell by left-clicking when its centroid falls within the ROI.
    10. Scroll through the z-stack to verify cell identity.
    11. Avoid counting the same cell more than once across multiple image planes.
    12. Count infiltrating cells separately if they contact or lie within the tumor boundary.
    13. Record infiltrating cells as a distinct category even when the cell centroid falls outside the ROI.
    14. Convert the ROI area from pixels to mm2.
    15. Obtain the pixel width, pixel height, and Z-step interval from Image > Properties.
    16. Calculation of normalized cell counts.
      1. Divide the raw cell count for each fish by the corresponding ROI area value.
      2. Record the resulting value as the normalized cell count.
      3. For absolute density measurements, calculate the physical area using the image calibration values obtained from Image > Properties.
      4. Divide the raw cell count by the calculated area (mm2).
      5. Record the result as cell density expressed as cells/mm2.

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Résultats

Le déroulement expérimental global est illustré dans la Figure 1. Des zebrafish adultes Tg(cd4-1:mCherry) ont été croisés avec des zebrafish Tg(dβh:MYCN;dβh:EGFP) afin de produire une descendance transgénique double dans laquelle les cellules CD4+ et les cellules exprimant dβh étaient marquées différemment par fluorescence mCherry et EGFP, respectivement. Les embryons ont été sélectionnés selon les marqueurs fluorescents appropriés, puis analysés soit pa...

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Discussion

Le protocole décrit ici utilise le poisson zèbre comme modèle in vivo pour visualiser et quantifier les cellules immunitaires au sein du microenvironnement tumoral (TME). Plusieurs facteurs sont essentiels à la mise en œuvre réussie de cette approche. Premièrement, le choix et la validation adéquats de lignées transgéniques de poissons zèbres composées sont indispensables. Les larves doivent être analysées pour détecter les profils d'expression fluorescente attendus et, si nécessaire, confirmées par génotypage. ...

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Déclarations de divulgation

Les auteurs déclarent qu’ils n’ont aucun intérêt concurrent.

Remerciements

Nous reconnaissons le soutien des National Institutes of Health (NIH ; 1UL1TR001430, CA215059 et NS140967), de l'American Cancer Society (RSG-17-204-01-TBG), de la National Science Foundation (1911253) et de la fondation Alex’s Lemonade Stand attribués à H.F. ; ainsi qu'une bourse Warren Alpert Distinguished Scholars attribuée à X.Q. Y.W. a bénéficié du programme de recherche pour étudiants de premier cycle de l'Université de Boston (UROP) et du programme de bourses d'été St. Baldrick’s. M.V. a été soutenu par le programme de bourses d'été St. Baldrick’s. Le contenu de cet article relève uniquement de la responsabilité des auteurs et ne représente pas nécessairement les points de vue officiels du NIH.

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Matériaux

Liste des matériaux utilisés dans cet article
NomEntrepriseNuméro de catalogueCommentaires
200 µL Gel Loading TipMilliporeSigma1022-0600Utilisé pour la manipulation des larves
Plantes artificiellesThe Hidden Reef62874200000000000Enrichissement optionnel pour la reproduction
Eau de JavelFisher Scientific50371500Utilisée pour la décontamination des embryons
Plugin Cell CounterNational Institutes of Health (NIH)N/APlugin Fiji utilisé pour la quantification cellulaire
Ordinateur et écranHP2TB68A8#ABAUtilisé pour l'acquisition et l'analyse d'images
Microscope confocalZeissLSM 710Utilisé pour l'imagerie des larves et des adultes
Moule cryogénique en plastiqueEpredia22-050-160Utilisé pour l'encapsulation dans le milieu OCT
CryostatThermo Fisher Scientific95-664-0EC70Utilisé pour la cryocoupe
Glace sècheFisher ScientificN/AUtilisée pour le transport des échantillons
Logiciel FijiNational Institutes of Health (NIH)Version 1.54pLogiciel d'analyse d'images
Filet fin en mailleCarolina Biological651345Utilisé pour la collecte des embryons
Incubateur pour poissonsVWR35960-056Maintenu à 28,5°C
PincesFisher Scientific10-316BUtilisées pour la manipulation des échantillons
Plaque à fond en verreIBIDI81218-200Utilisée pour l'imagerie
Agarose à bas point de fusionProlab Supply9012-36-6Utilisée pour le montage des larves et des adultes
Éclosionneur (réservoir principal, séparateur, section grillagée)Carolina Biological161937Utilisé pour la reproduction du poisson zèbre
Agitateur rotatifMidsciR2D-30Utilisé pour la fixation pendant l'incubation nocturne
Composé OCTTissue-Tek4583Milieu d'encapsulation pour la cryocoupe
Paraformaldéhyde (PFA)Sigma Aldrich158127-500GFixateur
Boîte de Pétri en plastiqueMilliporeSigmaBR452000Utilisée pour la manipulation des embryons et des larves
Solution saline tamponnée au phosphate (PBS)Gibco14080-055Utilisée pour les lavages et les procédures de fixation
Pipette Pasteur en plastiqueFisher Scientific50-998-100Utilisée pour le transfert des embryons et des larves
Lame de rasoirFisher Scientific12-640Utilisée lors de la préparation des échantillons
ConductimètreYSIEC300ACC-04Utilisé pour vérifier la salinité de l'eau des rotifères
RotifèresThe Hidden Reef116151603Utilisés pour l'élevage des larves
Thiosulfate de sodiumThermo Fisher ScientificAAA1762936Utilisé pour la neutralisation de l'eau de Javel
SaccharoseFisher ScientificS25590ARéactif de cryoprotection
Méthanesulfonate de tricaineWestern ChemicalNC0872873Anesthésique ; vendu par Fisher Scientific
PincettesFisher Scientific12-000-122Utilisées pour la manipulation des échantillons
Microscope à fluorescence droitLeicaM165Utilisé pour le criblage en fluorescence

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