Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

This method provides a way to quantify liver size in larval zebrafish. It also allows for assessment of the effects of genetic and pharmacologic manipulation on liver growth and development. This technique provides a rapid and precise way to visualize and measure the liver as well as the gut and pancreas.

In addition to quantifying liver size, this dissection method can be helpful for preparing the liver, gut, and pancreas for in situ hybridization or confocal imaging. Before attempting deskinning on experimental larvae, make sure you’ve spent ample time practicing on non-experimental samples and have a significant success rate. It is critical to visualize proper orientation and movement of zebrafish larvae and forceps in order to remove skin without disturbing surrounding organs.

At three to seven days post-fertilization, after euthanizing larvae, use a glass pipette and pipette pump to collect up to 15 larvae in a two milliliter tube. Wash larvae twice with one milliliter of cold 1X PBS on ice. Remove as much PBS as possible using a glass pipette and pipette pump and add one milliliter of cold four percent PFA in PBS.

Incubate at four degrees Celsius at least overnight with gentle rocking. Using a glass pipette and pipette pump, remove the PFA from the tube and rinse three times with one milliliter of cold PBS. Rock for five minutes in between rinses.

Pipette several larvae in PBS into one well of nine-well round-bottom glass dish. Under a microscope, remove the skin surrounding the liver using fine forceps to hold one larva on its back, gripping on either side of the head as gently as possible. Then use very fine forceps in the other hand to grab the skin just overlying the heart.

Pull skin down diagonally towards the tail of the fish in the bottom of the dish on the left or right side of the fish. Repeat for the other side. Continue grabbing flaps of skin and pulling down until all of the skin and melanophores overlying or near the liver have been removed.

If yolk is present for five to six days post-fertilization larvae, lift the yolk off in one piece by holding the fish with fine forceps on its back and using the very fine forceps to gently prod the yolk. For three to four DPF larvae, hold the fish with fine forceps on its back and use the very fine forceps to stroke the yolk starting from the ventral side. Scrape the yolk off in pieces.

Place the dissected larvae into fresh, cold PBS in a two milliliter tube using a glass pipette and pipette pump. To mount the larvae, pour a few milliliters of three percent methylcellulose onto the lid of clean, plastic Petri dish. Use a glass pipette and pipette pump to add larvae to the methylcellulose, adding as little PBS with the larvae as possible.

Under a dissecting microscope at low magnification, use fine forceps to orient the fish so they are laying on their right side facing left. Confirm that the fish to be photographed is aligned perfectly with one eye directly on top of the other eye. If the fish’s tail is bent, remove the tail by pinching it forcefully with forceps to remove it so that the fish lays flat.

Zoom in to high magnification and focus on the liver, making sure that the liver’s outline is clearly visible. Press the Capture Image button to snap a picture and save the file. Repeat for all fish, making sure the magnification is the same for each picture.

Take a picture of a micrometer using the same magnification. To use the program R to blind all liver pictures to avoid potential investigator bias and promote scientific rigor, rename files randomly and create a randomization file containing a list of the original file names and corresponding blinded file names. Open randomized files in order, starting with file one.

Choose the Freehand Selections tool and outline each liver. Press Control-M to measure the area of each liver. For any livers that cannot be accurately measured because they have residual skin, yolk, are out of focus, or are not intact, insert a placeholder measurement.

Save the measurements in a text file. To unblind and analyze data, open the measurements file and randomization file in a spreadsheet program. Insert a new column in the measurements file and add the original file names for the blinded files using the Randomization file.

Save this file as the Unblinded Measurements file. Sort the data by the original file name. Exclude any liver measurements for which pictures weren’t adequate.

If necessary, convert measurement values into the desired scale in square millimeters. Open the Scale bar in the image processing software. Use the Straight Line tool to measure one millimeter on the scale bar.

The image processing software then measures in the same units as the livers, giving a conversion factor. Use the conversion factor to convert measurements in the Unblinded Measurements file. Determine the mean and standard deviation and calculate P-values.

At six days post-fertilization, non-transgenic zebrafish larvae showed natural position and shape of liver overlying the gut. Transgenic zebrafish have significantly increased liver size as compared to their non-transgenic siblings. Some examples of inadequate images and micrometer are shown here.

Larva with skin covering the liver, larva with yolk obscuring the liver, larva with parts of the liver pinched off, and larva with liver dislocated and falling off, larva with missing liver, larva with liver outline that is difficult to identify, and larva with improper positioning. When attempting this procedure, it is important to use small and controlled movements to prevent damage to the liver. In addition to bright-field microscopy, we can also use larvae dissected in this way, for confocal microscopy, to visualize fluorescent reporter proteins or immuno-fluorescent staining, and for in situ hybridization.

The formaldehyde or PFA is an irritant and suspected carcinogen. Gloves should be worn when handling PFA and concentrated solutions should be handled in a chemical fume hood.