Peri-vitelline space (PVS) engraftment in 48hpf zebrafish larvae
note: 500*10^6 mL cells are injected instead of the normal 250*10^6/mL, this enhances overall samples viscosity and allows for better control of the injection site. PVP40 concentration can be adjusted upward to do the same
Injection:
- Pull glass capillary needles using “program #99”.
- Break needle to form an opening of app. ø20um (approximately 0,8-1,0 * the diameter of the injected cells).
- Carefully and thoroughly resuspend the cells using a 20µl pipette tip.
- Pipette cell suspension into the open glass capillary needle using a long (micro loader) tip.
- Affix the needle in to micro manipulator .
- Place app. 20 embryos on a 1% agarose dish, anesthetized in tricaine. (up to however many you can inject in a 20min timespan can be placed on a dish, just make sure the fish don’t dry out)
- Remove excess moisture to immobilize the fish using a 3 ml transfer pipette.
- Inject the embryos with approximately 50-100 cells into the peri-vitelline space. Modifying pneumatic pulse length and intensity on the Pico pump (start at app. 20Psi, 200ms and adjust accordingly).
- Ensuring that the embryos do not dry out, all embryos are injected.
- Injected embryos are flushed off with fresh egg water and transferred to a labeled clean Petri dish. (when the needle blocks you can increase the pressure (100-120psi) and use the “gated” setting to force the blockage through the needle )
- This process is repeated until all (or sufficient) embryos are injected.
p.s relatively large, stable pressure is required to successfully and repeatable move the cells through the needle opening, therefore a Pico pump combined with an external pressure line is generally used (instead of an injector with an internal compressor)