TITLE: Confocal imaging of live larval zebrafish for assessing peripheral neuropathy
AUTHORS: Sandra Rieger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol describes a method to mount live 8 day-old zebrafish larvae in agarose for capturing fluorescent transgene-expressing peripheral somatosensory axons with confocal imaging. We have previously described a similar method in the Journal of Visualized Experiments (1).</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bib24kcn.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">References: </span></div><div class = "text-block">1. G. S. O'Brien, S. Rieger, S. M. Martin, A. M. Cavanaugh, C. Portera-Cailliau, A. Sagasti, Twophoton axotomy and time-lapse confocal imaging in live zebrafish embryos. J Vis Exp, (2009). </div><div class = "text-block">2. S. Rieger, A. Sagasti, Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin. PLoS Biol 9, e1000621 (2011); published online EpubMay (10- PLBI-RA-9260R3 [pii] 10.1371/journal.pbio.1000621). </div><div class = "text-block">3. A. J. Pittman, M. Y. Law, C. B. Chien, Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points. Development 135, 2865-2871 (2008); published online EpubSep </div></div>

[STEPS]
?. Mounting of larvae for imagingBefore mounting, select fluorescent larvae under a fluorescent stereomicroscope. For this, add a few drops of tricaine stock to the dish in which the larvae are raised and wait until larvae are anesthetized. Assess anesthesia by touching larvae with a pipette tip. If no response is observed, transfer fluorescent larvae into small petri dish containing the tricaine working solution. With a glass Pasteur pipette, transfer anesthetized larvae (one at a time) from the petri dish into a 1.5 ml tube containing 1.2 % of the low-melt agarose (keep at 42ºC in a heat block prior to use to maintain as a liquid). Transfer a drop of the agarose containing the larva into an imaging chamber. Immediately position the larva on its side for imaging from the lateral view and on its back to image from the dorsal view. Immobilized embryos are then immersed in tricaine working solution and the chamber is covered with a lid (if using the glass bottom dish) or sealed with a frosted slide following the application of silicon grease to the top of the Teflon ring (when using the Teflon chamber apparatus). Do not leave air bubbles in the imaging chamber after it is sealed, as they will interfere with imaging in the bright field mode.
?. Confocal imagingFollowing positioning of the mounted larvae on the inverted microscope, utilize a PlanNeofluar 20x/0.50 (Zeiss) air objective and a 1.6x digital zoom to capture the GFP-expressing axons with a 473 nm diode laser. To record stacks, capture 3 µm sections according to the owner’s manual. Project each stack into a single image. The projected images can be exported as tiff files and processed in Adobe Photoshop for publication. Alternatively, load stacks into Imaris imaging software and process with Filament tracer module to quantify features of axon degeneration such as axon debris and axon density.
?. Potential Pitfalls:1. Larvae die during the mounting or imaging procedure: a) If larvae die, the agarose may be too warm, which is typically the case if the agarose is prepared immediately prior to mounting of the larvae. In this case, let agarose cool down for sufficient amount of time (at least 30 minutes). b) The tricaine concentration may be too high, which negatively affects larval health. Also, re-test the pH of the tricaine, which should be pH 7. c) Larvae are unhealthy due to growth in the medium or lack of food. The larvae typically need to be fed with larval diet after 5 dpf due to the depletion of the yolk. 2. Images on the confocal microscope are blurry. This could be due to a dirty objective or excessive opening of the pinhole.