Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

This method can help detect the bone toxicity induced by chemicals in zebrafish. Alizarin Red Staining of fixed tissue generates a permanent record of skeletal changes that can facilitate comparisons of several specimens. This technique could be helpful to study osteoporosis in the zebrafish model, since one of the characteristics of osteoporosis is the decrease in bone mass.

Remove 10 zebrafish larva randomly from each group at nine days post fertilization, and fix the fish in one milliliter of 2%paraform aldehyde and 1X PBS for two hours. Decant the solution and wash the zebrafish larva using 100 millimolar Tris HCL with a pH of 7.5 or 10 millimolar Magnesium Chloride. For de-staining, decant the solution and incubate the larva for five minutes in a series of solutions as described in the manuscript.

After the final incubation, decant the solution and remove the pigment by bleaching with a solution consisting of 3%hydrogen peroxide and 0.5%potassium hydroxide. Observe once every 10 minutes until the pigment is completely removed. Rinse the zebrafish larva several times with 25%glycerin, 0.1%potassium hydroxide for 10 minutes each until there are no bubbles.

Stain in the larva by soaking in one milliliter of 0.01%Alizarin. Decant the solution, and add one milliliter of 50%glycerin or 0.1%potassium hydroxide to clear the background. Store the fish in fresh 50%glycerin or 0.1%potassium hydroxide for subsequent observation.

Transfer one larva at a time onto the glass slide keeping the larva in the middle of the liquid drop. Observe the larva under a stereo microscope. Turn on the camera.

Open the software, and keep the default settings. Click on AE and choose an appropriate exposure time to obtain the best image. Capture all the images under the same settings.

Save the images in dot tiff format for later analysis. Double click the imageJ icon. To analyze the saved images, click on file, then open, then click on image, then type and select eight-bit.

Click on edit, and invert. Click on analyze, then calibrate. Select Uncalibrated OD in the pop-up interface.

Check global calibration at the bottom left of the lower interface, and click okay. Click on analyze, set scale, then remove scale. Check global below, and click okay.

Click on analyze, then set measurements. Select the item area in the pop-up interface. Check the limit to threshold below to measure only the selected range, and click okay.

Click on image, adjust, then threshold, and slide the slider in the middle of the pop-up interface. To select the appropriate threshold so that all targets to be tested in one image are selected. And click set.

Click on analyze, then measure, save the dates. Alizarin Red Staining is a sensitive and specific method for measuring changes in bone mineralization in zebrafish larvae. At nine days post fertilization, many bones of the head skeleton such as parasphenoid, opercular, ceratobranchial, and notochord are mineralized and therefore stained in red.

In contrast, non-bony structures like otoliths, appear brown black. Digital analysis performed to quantify the total stained area, showed a significant decrease in the stained area for the 10 and 20 milligrams per liter of lead acetate treatment groups. The changes in bone mineralization showed dose dependency.

For better staining effect and observation, it's important to remove the original pigment of zebrafish larvae, and remove the bubbles after bleaching.