Versatile Dual Mounting Enables Larval Zebrafish Imaging Across Microscope Configurations

Materials and reagents

Biological materials

1. Tg(erbb3b:gal4)^{nkgsaizGFFD37A}, Tg(UAS:eGFP)^nkuasgfp1a, and Tg(olig2:dsRed2)^vu19 zebrafish transgenic lines were used for mounting protocol validation [9,10].

Reagents

1. Instant Ocean salt (Instant Ocean, catalog number: 77780); store in an airtight container in a cool, dry place at room temperature (RT)

2. 3-Aminobenzoic acid ester (tricaine) (Pentair, catalog number: TRS1); store in an airtight container in a cool, dry place at RT

3. Tris (Tris[hydroxymethyl]aminomethane or Trimethamine) (Bio-Rad, catalog number: 1610716); store in an airtight container at RT

4. 1-Phenyl-2-thiourea (PTU) (Sigma, catalog number: P7629, CAS number: 103-85-5); store in an airtight container at RT, protected from light

5. Agarose, low gelling temperature (Sigma, catalog number: A94114); store in an airtight container at RT

Solutions

1. Egg water (see Recipes)

2. PTU stock solution (10×) (see Recipes)

3. PTU water (see Recipes)

4. Buffered tricaine methanesulfonate (MS-222) stock solution (see Recipes)

5. Anesthetic solution (see Recipes)

6. 0.8% low-gelling agarose (see Recipes)

Recipes

1. Egg water

2. PTU stock solution (10×)

3. PTU water

4. MS-222 stock solution

5. Anesthetic solution

6. 0.8% low-gelling agarose

Laboratory supplies

1. Breeding tanks (Aquaneering, catalog number: ZHCT100)

2. Dumont Tweezers, pattern #5; 0.10 × 0.06 mm tip size (Roboz Surgical, catalog number: RS-5065)

3. 100 × 15 mm Petri dish (Falcon, catalog number: 351029)

4. 35 mm glass-bottom confocal dish, non-treated (Avantor, catalog number: 75856-746)

5. 60 mm Petri dish (Fisher, catalog number: FB0875713)

6. Disposable glass Pasteur pipettes (VWR, catalog number: 53283-916)

7. Pipette pump, 10 mL (Honbay, ASIN: B07P61YDXB)

8. Micro dissecting needle holder, 4 3/4’’ (Roboz Surgical, catalog number: RS-6061)

9. Insect pins 0.30 mm size 00 (Roboz Surgical, catalog number: RS-6081-30)

10. Glass microscope slides (Sail Brand, catalog number: 7105)

11. 50 mL borosilicate glass beaker (Maccx, catalog number: BKL050-012)

12. Borosilicate glass test tubes with autoclavable caps (ULAB, catalog number: UTT1009)

13. Petroleum jelly (Vaseline, catalog number: UNI34500)

14. Super glue (Krazy Glue, catalog number: KG58548R)

15. Wood applicator stick, 6” × 1/12” (Oxford Instruments, catalog number: ID 51-1625-0580)

16. Zeiss immersion W oil 2010 (Microscope World, catalog number: 444969-0000-000)

Equipment

1. Fisherbrand Basic 180 L Gravity Incubator (ThermoScientific)

2. ECLIPSE Ti2 (Nikon) equipped with a Dragonfly 200 high-speed confocal platform (Andor Oxford Instruments), a 40×/1.15 W APO LWD numerical aperture water immersion objective (Nikon), and a motorized stage (Applied Science Information)

3. Air-MP (Nikon), equipped with a 25×/1.10 W numerical aperture water immersion objective (Nikon), ProScan III motorized stage (Prior Scientific), Chameleon Vision II laser system (Coherent), and an HEC series Thermo-con cooling system (SMC)

Software and datasets

1. Fusion 2.6.0 (Andor Technology; 2026, license required)

2. Nikon Imaging Software, Elements Advanced Research 5.21.03 (Nikon; 2020, license required)

3. Imaris 11.0 (released 2025; license required)

Procedure

A. Obtain zebrafish embryos

Note: This section details the process of gathering embryos for use in microscopy experiments.

1. In the afternoon, prepare breeding tanks with a desired female-to-male ratio of 2:2.

2. The following morning, collect and store the freshly laid embryos in a Petri dish filled with egg water.

3. Rear embryos in a 28.7 °C incubator, replacing egg water with PTU at 24 h post-fertilization (hpf) to prevent the formation of pigmentation.

4. Manually dechorionate embryos with fine tip tweezers as needed by 48 hpf to preserve trunk linearity.

5. Keep embryos at 28.7 °C until the desired imaging time point, to ensure proper developmental pace [11].

B. Create mounting model

Note: This section outlines the creation of the dual-configuration mounting model for use across orientations with traditional mounting.

1. For compatibility with a standard microscope stage, use super glue to adhere the edge of a 60 mm Petri dish lid to a glass microscope slide (Figure 1). Let it cure for at least 10 min before use.

Figure 1. Assembly of the 60 mm Petri dish lid and glass slide (left) with super glue application along the dashed yellow lines (right)

Note: Modeling clay can be used as an alternative adaptation, ensuring that the Petri dish lies both as flat and as close to the glass slide as possible (Figure 2). However, if the microscope has an adjustable stage holder, the use of a glass slide is not necessary, and the 60 mm Petri dish lid can be directly accommodated.

Figure 2. Alternative adherence of 60 mm Petri lid edges to a glass slide using modeling clay

2. Either by hand or with a wood applicator stick, line the rim of a 35 mm glass-bottom imaging dish to stamp a circle onto the 60 mm Petri dish lid (Figure 3). This will serve as a placement guide and reinforce the intended hydrophobic seal.

Figure 3. Mounting model and imaging dish (left) used to create a petroleum jelly reference stamp on the Petri dish lid (right)

C. Traditional mounting of embryos

Note: Here, the traditional mounting approach is detailed for the lateral orientation of zebrafish embryos.

1. Place embryos in a 4% anesthetic solution [12] for 5–10 min to ensure samples are immobilized.

2. Melt 0.8% low-gelling point agarose by heating the glass test tube in a water bath using a 50 mL beaker, microwaving in 15-s intervals.

3. Allow the agarose to cool until the glass tube can be held comfortably by hand but remains liquid.

4. Using a glass pipette attached to a pipette pump, transfer embryos into the cooled agarose and then to a 35 mm glass-bottom imaging dish.

5. Gently manipulate the embryos to a lateral orientation in the liquid agarose with a dissecting needle.

Note: Ensure that samples are flat against the glass surface to minimize the necessary working distance for imaging.

6. Allow agarose to set for 10–15 min.

7. Fill the 35 mm glass-bottom imaging dish to the very top with anesthetic solution.

D. Assemble with the mounting model

Note: The combination of the traditional mounting approach with the dual-configuration mounting model is outlined in this section.

1. Either by hand or with a wood applicator stick, line the rim of the 35 mm glass-bottom imaging dish with a thick layer of petroleum jelly (Figure 4).

Figure 4. Glass-bottom imaging dish (left) lined with petroleum jelly (right)

2. Carefully fill the petroleum jelly–lined imaging dish to the very top with anesthetic solution.

3. Invert the dual-configuration mounting model 180° and align the petroleum ring with the petroleum jelly–lined confocal dish below. Press together to establish a hydrophobic seal (Figure 5).

Note: If there is a large air bubble, unseal the 35 mm glass-bottom confocal dish and fill with anesthetic solution until a convex meniscus forms. Then, reseal the mounting model.

Figure 5. Placing the mounting model onto the imaging dish lined with petroleum jelly (left) to establish a hydrophobic seal (right)

4. Reinvert the mounting model (Figure 6).

Figure 6. Mounting model ready for use on an upright microscope stage

Validation of protocol

This protocol has been routinely validated by use in our lab to conduct two-photon laser ablation experiments on an upright microscope, followed immediately by time-lapse imaging on an inverted spinning disk confocal (Figure 7). This approach enables rapid switching between imaging modalities, minimizing the time during which the effects of laser perturbation go unobserved. This capability is particularly important for visualizing debris clearance kinetics, allowing for cellular dynamics to be monitored closely from the moment of injury onward. The hydrophobic seal effectively maintains sample accessibility and stability across configurations, contributing to ease in re-identifying the same anatomical region across microscopes (Figure 8).

Figure 7. Mounting model demonstrated on upright two-photon (left) and inverted spinning disk confocal (right)

Figure 8. Protocol validation using Tg(erbb3b:gal4)^{nkgsaizGFFD37A}; Tg(UAS:eGFP)^nkuasgfp1a; Tg(olig2:dsRed2)^vu19 larval embryos at 3 days post-fertilization (dpf). Only the GFP channel is shown in panels A and A’. To explore the phagocytic potential of motor exit point (MEP) glia, an erbb3b⁺ cell (A) along the caudal primary spinal motor neuron pathway was targeted with laser ablation (A’), as indicated by the yellow circle, using an upright two-photon microscope [13]. This anatomical region of interest was time-lapsed immediately after on an inverted spinning disk confocal (B). This approach allows for targeted focal ablation on a multiphoton microscope with limited red wavelength access, followed by faster imaging speeds and red channel accessibility on a spinning disk confocal. The confocal image in B was pseudocolored in Imaris, with dsRed2 in green and GFP in pink.

General notes and troubleshooting

General notes

While this mounting strategy offers flexibility across imaging configurations and is broadly compatible with common microscope stages, consistent repositioning of the same region of interest still requires visual landmarks like the yolk extension. This model does not change working distance requirements; however, users should still confirm appropriate clearance under upright objectives per the standard procedure.

Troubleshooting

Problem 1: A single air bubble is trapped in the 35 mm imaging dish, obscuring the sample.

Cause: Insufficient volume of anesthetic solution can cause trapped air upon hydrophobic seal.

Solution: Break the hydrophobic seal and fill the dish to maximum capacity, aiming for a convex meniscus with anesthetic solution prior to resealing.

Problem 2: Adhesive degradation between the Petri dish lid and glass slide.

Cause: Standard cyanoacrylate (super glue) forms a semi-permanent bond with glass, so re-adherence is often necessary over time.

Solutions: Re-adhere the Petri dish lid to the glass slide with super glue. Alternatively, more durable materials like UV-curable resin or modeling clay can be used. With the latter, take care to ensure the slide is as flat and flush against the dish as possible.

Acknowledgments

Conceptualization, H.M., L.F.; Investigation, H.M.; Writing—Original Draft, H.M.; Writing—Review & Editing, L.F.; Funding acquisition, L.F.; Supervision, L.F. Funding sources that supported the work: Jupiter Life Science Initiative, Stiles-Nicholson Brain Institute.

We thank the Fontenas lab members for their valuable input on this project and MacKenzie Tackett for great fish care.

The following figures were created using BioRender: Graphical overview, Murphy, H. (2026). https://BioRender.com/x579fyx.

Competing interests

The authors declare no conflicts of interest.

Ethical considerations

Husbandry and generation of embryos: All animal studies were approved by the Florida Atlantic University (FAU) Animal Care and Use Committee. Embryos were raised at 28.7 °C in egg water. Zebrafish cannot be sexed until adulthood; therefore, embryos of undetermined sex were used. Pigmentation was inhibited in embryos using phenyl-thiourea (PTU) (0.003%) in egg water. All studies were conducted using stable, germline transgenic lines. Fish were housed and maintained in our fish facility at FAU under standard laboratory conditions. Adult zebrafish strains were kept at 28 °C on a 14/10 h light/dark cycle. To generate embryos, adult male and female zebrafish were housed in breeding tanks overnight and allowed to spawn naturally.

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