Zebrafish Xenograft Assay: A Method to Assess the Anti-cancer Efficacy of Therapeutics on Tumorigenicity in Zebrafish In Vivo

- First, place the eggs into a Petri plate, and add Danieau's solution to maintain freshly collected eggs. Add phenyl thiourea to inhibit pigmentation and incubate for a few days. After incubation, dechorionate the embryos using sharp forceps and let them grow further.

Next, seed the desired number of cancer cells in a culture flask and incubate overnight. Now add anticancer compounds and further incubate. After incubation, add fluorescent cell tracker dye, which will bind to the cell membrane and stain the cells.

Next, add trypsin for cell dissociation and dilute the cells in the phenol red PBS solution before the procedure to assess pH. Mount anaesthetized zebrafish embryos on an agar plate. Now load the microcapillary with cancer cell suspension and inject it into the yolk sac of embryos.

Transfer embryos to culture plates containing Danieau's solution and incubate further. Next, transfer a few embryos on a glass slide and add methylcellulose to immobilize them. Place the slide under a fluorescence microscope and quantify the tumor size using the software.

Anticancer compounds usually inhibit the tumor forming capacity of cancer cells. In the following protocol, we will study th