Molecular Vision: Gonzalez-Fernandez, Mol Vis 2011; 17:2956-2969. Table 1

Table 1. Trouble shooting guide

Category	Problem	Recommendation
Embryo handling	Embryos damaged during insertion into the agarose foxholes.	Add 0.05% food coloring to the agarose to improve visualization of the holes. Tip: touch closed forceps to cement gland, and drag embryo over hole. The gland will adhere lightly to the forceps. Then, push embryo backward guiding its posterior end into the hole with a second forceps (not illustrated in video)
	Spontaneous movements interfere with foxhole insertion and microinjection.	Add 0.01% benzocaine to bath (a 0.05% stock keeps for at least one month at 4 °C). (Note: MS-222, a commonly used fish and amphibian anesthetic, impairs visual pigment regeneration by binding 11-cis retinal [40], which presumably is responsible for its toxicity [41]. For these reasons, we prefer benzocaine.
	Embryos damaged during removal from foxholes.	Embryos are extremely delicate! Never grab. Once in the hole, the strength of the cement gland adhesion is insufficient to pull the embryo out. The best method is to use a large bore plastic, or fire-polished glass pipette. Position the pipette over the hole lightly touching the agarose and apply gentle suction. The embryo will readily pop out, and the hole can be reused for another embryo.
	Uncertain that material is actually being injected.	Addition of colored or fluorescent dye is helpful in confirming the injection. 0.025 - 0.05% phenol red is convenient. Alternatively fluorescent dextran or beads can be used. Successful injections can be confirmed by visualizing the embryo in vivo under a fluorescent stereomicroscope, and in histological sections. Adjust the backpressure so that a gentle flow of material continually emerges from the tip. If the backpressure is insufficient, water will flow into the pipette diluting the material intended for injection. Discard embryos that do not show typical pattern of ventricular/optic vesicle filling (see Figure 3, Figure 6, and Figure 9).
	Insufficient number of embryos injected	The injection window is finite - stay organized! The day before, check that enough nitrogen gas and micropipettes are available. Set up multiple injection stations. Rear embryos at various reduced temperatures (14 – 20 °C) to widen window of specific stage availability. Alternatively, carry out in vitro fertilizations at several points during the day.
Micropipette problems	Difficulty filling pipette from limited supply of valuable injection material.	Under a stereomicroscope lower by hand, or with the aide of a micromanipulator, the micropipette tip almost horizontally into a droplet on a Parafilm rectangle. While carefully ensuring that the tip remains within the drop, start suction. Do not remove the tip until the vacuum has finished.
	Difficulty in positioning micropipette	A micromanipulator can be helpful. However, we find that it is more efficient to hold the micropipette directly by its handle. Rest fingers on microscope stage, and keep the glass pipette short (~1 cm) to minimize hand vibrations.
Injections Photography and Tissue Processing	No ventricle or optic vesicle fill observed. Optic vesicle not filling despite brain ventricle filling. Injected material not confined to cerebral ventricles, or optic vesicle. Poor contrast of photographed embryos Spontaneous drifting of embryo during photography Fixation and sectioning are laborious and time consuming Fluorescence not visible in sections cut from paraffin embedded embryos.	Generally due to tip obstruction. Clogs occur from tissue or agarose stuck in the tip and/or drying of test solution when pipette is taken out of the bath for example between dishes. The tip can be broken to restart the flow. Avoid leaving the pipette out of water for any extended time. A gentle dye flow from the pipette tip is a good sign that the tip is not clogged. Separation of optic vesicle and neural tube lumens may have occurred. Use younger embryos (< stage 31). Penetration too deep, or injection pressure too high. Note that the diencephalic ventricle is just under the frontal ectoderm (see Figure 3). To enter the ventricle, it is therefore only necessary to puncture through the thin ectoderm and proencephalon. Do not overfill. Find the maximum pressure and time settings that do not cause visible inflation of the optic vesicles during the injection. Use fiber optic light source angled ~30 ° from one side. Living embryos drift forward. One solution is to formalin fix before photographing. For living embryos, lay broken coverslip pieces in front of embryo to prevent movement. Tissue processing and sectioning of multiple embryos is often the most challenging part of any experiment. Fixation and sucrose/OCT infiltration are efficiently performed in 24-well cell culture plates. To orient multiple embryos so that they can be simultaneously sectioned through their eyes, align four to six embryos lying down in the embedding mold so that their anterior ends all touch the side of mold. Move the embryos within the viscous OCT with a metal needle. Taking care not to disturb their alignment, freeze them in an isopentane bath over liquid nitrogen. Working quickly cut out the aligned embryos in a single rectangular block. Re-embed the block in a heads down orientation in a fresh mold prefilled with OCT and freeze. In this way, the alignment is exact, allowing the eyes from all embryos to be included in same histological section. The xylene treatment required for tissue infiltration with paraffin is not compatible with the Cy3-FluoSpheres®. Embed in methacrylate resin (Figure 7), or perform frozen sections (Figure 11).