Dissection of Zebrafish Craniofacial Tissues Upon Staining with Alcian Blue

We are exploring the mechanisms by which zebra fish palate grows over time using fixed samples as well as time lapse imaging of embryos with high spatial temporal resolution using light sheet microscopy. Our protocol is simple and easy to follow. It lays down detailed steps to dissect out, and separate craniofacial cartilages of a five-day old zebrafish larva.

This protocol has enabled careful characterization of palate shape at tissue and cellular scales, ultimately allowing us to assess the effect of commentary antigens on palate morphogenesis. To begin, obtain 20 to 25 anesthetized zebra fish larvae, and remove the anesthetic once the larvae have stopped moving. Add 1.5 milliliters of 4%paraform aldehyde to the tube, and place the sample on a rocker set to 60 revolutions per minute at room temperature for two hours.

Using a pipette, remove as much of the fixative as possible from the tube. Then add 1.5 milliliters of 50%ethanol to the sample, and place it on a rocker at room temperature set to 60 revolutions per minute for 10 minutes. Now using a pipette, aspirate the ethanol from the tube, and add 1.5 milliliters of alcian blue stain solution to the sample.

Incubate the larvae in the stain solution for 18 to 20 hours at room temperature while rocking at 60 revolutions per minute. Next, remove the stain solution using a pipette, and wash the larvae with 1.5 milliliters of distilled water for one to two minutes on a rocker at room temperature. Then add one milliliter of bleach solution to the sample, and incubate at room temperature for 20 minutes without covering the tubes.

Using a pipette, remove the bleach solution from the sample, and treat the sample with tissue clearing solutions one and two. After removing solution two, add 1.5 to two milliliters of storage solution to the sample. Due to the glycerol content, observe as the stained samples gradually sink to the bottom within 10 minutes.

Place a stained larva on its lateral side in the agarose coated Petri dish. Using forceps, gently scrape off the yolk from the body of the larvae. After yolk removal, transfer the larvae to a fresh Petri dish to avoid interference during dissection, hold the larvae near the posterior of the head using one pair of forceps.

With another pair, carefully remove the eyes without damaging the surrounding cranial facial structures. While holding the larva, still with one pair of forceps, make an incision at the center of the tissue dorsal to the neuro cranium. Then pinch and pull off the brain, and associated tissues.

Next, separate the head of the larvae from the rest of the body. The resulting head region should include the neuro cranium, and visceral cranium still attached at the anterior and posterior ends. Carefully sever the two connecting points between the neuro cranium, and visceral cranium with forceps to fully separate them.

Finally, place one or two drops of 100%glycerol on a clean glass slide, and transfer the dissected neuro cranium or visceral cranium to the slide. Using forceps, gently place a cover slip on top of the tissues ensuring no air bubbles are trapped, and seal the cover slip with nail polish before imaging. The dissected neuro cranium displayed multiple clearly labeled structures, including the ethmoid plate, trabecula, and pericardial cartilage, enabling detailed structural analysis.

The visceral cranium included Meckel's cartilage, and additional elements such as the palato quadrate, and basilbranchial cartilages. Alcian blue staining at five days post fertilization enabled clear visualization of the ethmoid plate. The width and length of the ethmoid plate were consistently measurable using image analysis, and the length was significantly greater than the width, and quantified data from 15 samples.

The ethmoid plate area was extracted from image data, and confirmed through quantification with clear boundary markings. Distinct cellular arrangements were observed in the ethmoid plate with cuboidal cells in the medial region, and columer cells in the lateral parts. Dappy staining allowed visualization of individual cell nuclei within the ethmoid plate.