A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

1. Preparation of Solutions and Pulled Glass Needle

1. Make a 25x stock solution of Tricaine (3-amino benzoic acid ethyl ester, also called ethyl 3-aminobenzoate) at 4 mg/mL in 20 mM Tris pH 8.8 and bring that solution is at pH 7. Aliquot by 4 mL and store at -20 °C.
   NOTE: The anesthetic Tricaine acts preferentially on neural voltage-gated sodium channels thereby blocking muscle twitching and movement⁶.
2. Make a working solution of Tricaine at a final concentration of 0.17 mg/ml in E3 embryo medium.⁷
   NOTE: Make the Tricaine working solution extemporaneously as the pH of this solution drifts.
3. Dissolve the low melting point agarose to a final concentration of 1.5% in E3 embryo medium in a 50 mL tube by heating the solution in a microwave. Let this solution equilibrate to 42 – 45 °C in either a water bath or bench-top incubator. If preparing large dishes for vertical light-sheet imaging (step 4), make an additional 25 mL of 1% standard melting agarose in E3 embryo medium.
4. Use borosilicate glass with filament capillaries with dimensions of OD 1.20 mm, ID 0.69 mm, length 10 mm.
5. If capillaries are pulled on the type of heating-filament needle puller described in the Table of Equipment and Reagents, use the following settings: Heat 600, Pull 120, Velocity 50, Time 225, Pressure 500. With a pair of sharp forceps, break the needle just past the point at which it flexes to create a clean and sharp needle for orientating embryos and removing excess agarose. A micro-scalpel can also be used to remove excess agarose.

2. Embedding of Embryos for Inverted or Upright Microscopy

1. Raise the embryos up to the appropriate stage in E3 embryo medium.⁷
2. At the required stage, dechorionate embryos with a pair of sharp forceps underneath a binocular dissecting microscope.
3. Incubate the dechorionated embryos for at least 5 min in the Tricaine working solution.
4. Once the melted agarose solution has cooled to 45 °C, use a glass Pasteur pipette to transfer the dechorionated embryo directly into the 50 ml tube with minimal transfer of E3 medium.
5. Remove embryo together with approximately 1 ml of mounting medium and add approx. 100 µl of mounting medium together with the embryo to the central circle of a 35 mm glass-bottomed petri dish with a 10 mm microwell.
6. As the mounting medium is setting, move the embryo to the edge of the circle of agarose with the tail facing outwards (Figure 1A). Use the capillary needle to maintain the embryo in the desired lateral orientation until the gel is completely set. To image posterior body development, take care to orientate the embryo in as lateral an orientation as possible. Therefore, maintain the embryo in this position by careful adjustments with the capillary needle.
   NOTE: Multiple embryos can be added to the mounting dish at this point, if required. If this is the case, transfer the low-melt agarose into the dish first and add the embryos to the center of the agarose drop. Then, push the embryos out to the edges of the glass ring and orientate into the desired position. Up to six embryos can be mounted this way before the agarose becomes solidified.

3. Removal of Excess Agarose Around the Posterior Body

NOTE: This section describes the procedure by which agarose is removed from the region surrounding the posterior body. In the case of posterior body elongation, it is important to ensure that the tail can grow-out normally. By removing the agarose after the embryo has been completely included by agarose, the embryo is left enclosed by the head region and approximately half of the yolk sac.

1. Once the agarose drop has set, flood the petri dish with the Tricaine working solution.
2. Under a dissecting binocular microscope with a transmitted-light base, adjust the mirror position and angle of incident light such that the strong contrast allows for the cuts in the agarose to be seen clearly through the operation.
3. To perform cut 1 (Figure 1), use the capillary needle or the micro-scalpel to cut the agarose mid-way along the yolk, from a position just posterior to the forming heart field to the position of the somite 5 (the 5^th anterior somite).
4. Start the initial cut adjacent to the forming heart as shown, and the full way through the agarose to the glass.
5. Keep the capillary or micro-scalpel deep within the agarose, and slowly saw up and down while beginning to make a long cut over the embryo as shown Figure 1. Cut as close to the embryo as possible without puncturing the embryo or yolk.
6. Next, make cuts 2 and 3 (Figure 1), starting from the position against the embryo and with the cut going all the way down to the glass bottomed dish. Make cut 2 so that it is tangential to the first 5 somites, allowing for full dorsalward unfolding of the posterior body. Make cut 3 at a 90° angle with the yolk surface and starting from the heartfield.
   NOTE: Cutting the agarose until the edge of the glass circle (as shown in Figure 1) aids in removing the agarose as a complete block (step 3.6). However, this is not necessary.
7. Starting at the intersection of cuts 1 and 3, make a slow diagonal cut towards the end of cut 2 whilst slowly lifting upwards in order to dislodge the square of agarose surrounding the posterior body.
   Notes: In some cases, this will be released in one block and the operation is completed in one go. In others, it may take several attempts to dis-lodge all the agarose surrounding the embryo.
8. With a pair of sharp forceps, remove the dislodged blocks of agarose from the embryo medium. To aid in this process, move the agarose pieces to the side of the dish and use the wall of the petri dish as support whilst lifting out the agarose pieces.

Figure 1: Diagrams of Mounting Set-ups. (A) The diagram shows the position of the mounted embryo within the center glass ring of a petri dish. On the right is a zoom of the embryo with each successive cut through the surrounding agarose shown with dottted red lines. (B) The mounted embryos is diagrammed in lateral view displaying the ease of access for both inverted and upright objectives. (C) A similar digram showing how embryos can be mounted for vertical light sheet imaging set-ups. Please click here to view a larger version of this figure.

9. If muscle twitching is not completely blocked at that point, add drops of Tricaine from the stock solution of 4 mg/ml, pH 7.

4. Mounting of Embryos for Vertical Light-sheet Microscopy

NOTE: This is a variation on the method outlined above that allows for the access of multiple objectives for imaging of samples by vertically-orientated SPIM. The idea behind this variation is to lift the sample slightly higher than the bottom of the dish, to allow for easy access of two imaging objectives.

1. Prior to sample mounting, coat a 100 mm plastic petri dish with 1% agarose in E3 medium to a height of 5 mm and allow to set.
2. Place a drop of 1 ml low-melting point agarose to the center of the dish and allow to set.
3. Embed embryos in low-melting point agarose as in section 2. However, this time, remove the embryo from the 50 ml tube of agarose with a smaller drop of solution (0.5 ml) and place this small drop on top of the 1 ml drop in the center of the dish.
4. Use the capillary needle to place the embryo in the center of the small drop and maintain its correct orientation until gel is set.
5. Flood the dish with Tricaine working solution and remove the excess agarose as in section 3 but without cutting through the cushion of 1% standard agarose.