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Preprint

Tracer injections

NO Natasha O'Brown
SM Sean G Megason
CG Chenghua Gu

Last updated date: Sep 12, 2025 Forks: 0

original An abbreviated version of this protocol was published in eLife in Aug, 2019
Suppression of transcytosis regulates zebrafish blood-brain barrier function
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  1. Load the needle with a few uls of whatever you’re injecting.
    • For fluorescent tracers, I usually start with 5 mg/ml concentration of tracer and add my phenol red as I would for single cell injections to see where things go

  2. Place the larvae upside down in agarose injection molds (we now use these 3D printed molds: thingiverse.com/thing:4968318), with their hearts facing up. I like to have the higher edge of the mold on the opposite side of where there needle is coming from.

  3. Gently pierce the cardiac sac, and inject with 2 nl of solution. The sac should visibly fill with a reddish tint if using the phenol red. Then retract the needle. Hearts may momentarily stop beating, but should resume in less than a minute.
  4. Can visibly see the tracers in the skin on the epifluorescent scope in less than 5 minutes
  5. Measure tracer intensity in the brain parenchyma 1 hour later and normalize to tracer intensity in the cranial blood vessels
Copyright: © 2025 The Author(s); This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. O'Brown, N, Megason, S and Gu, C(2025). Tracer injections. Bio-protocol Preprint. bio-protocol.org/prep2857.
  2. O'Brown, N. M., Megason, S. G. and Gu, C.(2019). Suppression of transcytosis regulates zebrafish blood-brain barrier function. eLife. DOI: 10.7554/eLife.47326

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