Feb 23, 2024

Preparation of JF dye for retro-orbital injection in mice V.2

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm7rznv3p/v2
  • 1HHMI Janelia Research Campus
Boaz Mohar
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm7rznv3p/v2
Protocol Citation: Boaz Mohar 2024. Preparation of JF dye for retro-orbital injection in mice. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm7rznv3p/v2Version created by Boaz Mohar
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working. Changed 200ul total to 100ul. If need to do 200ul (200nmol dye), it is done with 100ul per eye
Created: February 23, 2024
Last Modified: February 23, 2024
Protocol  Integer ID: 95629
Keywords: orbital injection in mice, preparation of jf dye, retro orbital injection, jf dye, orbital injection, dye, injection, mice
Funders Acknowledgements:
HHMI
Abstract
How to prepare JF dyes for retro orbital injection.
Materials
STEP MATERIALS
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
Pluronic™ F-127 (20% Solution in DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3000MP
PBSInvitrogen - Thermo Fisher
29G x 12.7mm 0.3ml syringeUlticareCatalog #09230
Protocol materials
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
Pluronic™ F-127 (20% Solution in DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3000MP
PBSInvitrogen - Thermo Fisher
29G x 12.7mm 0.3ml syringeUlticareCatalog #09230
Dye preperation
Take an aliqote with 100 Mass Percent of dye from -20 °C storage.


Add 20 µL DMSO and do ~10 up and down with the pipeter


Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650


Add 20ul Pluronic F-127 20% in DMSO ~10 up and down with the pipeter


Pluronic™ F-127 (20% Solution in DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3000MP


Add 60ul 1x PBS ~10 up and down with the pipeter
PBSInvitrogen - Thermo Fisher

Drew up to 100ul without bubbles to a 29G syringe
29G x 12.7mm 0.3ml syringeUlticareCatalog #09230



Follow the Retro-orbital injection protocol
Protocol
NAME
Retro-orbital injection of virus or dye in mice
CREATED BY
Boaz Mohar

Prepare either virus dilution (5.0e11-1.0e12) with PBS prepare or a dye aliquot. Injection is up to 200ul per mouse.



Take one mouse from the cage into an induction chamber and start isoflurane at 3% with flow rate of ~1L/min
After the mouse has slow breathing (~1/s) move the mouse to a nose cone delivery of isoflurane at 1.5% with around the same flow rate.
Expose the eye ball with two fingers (above and below) and make sure the you are not pressing on the trachea and the mouse's breathing is regular.
Use a 29G syringe at a 30-degree angle beveled downwards until you hit the bone, retract a bit and slowly inject. See cited paper for more details.
Citation
Tal Yardeni, Michael Eckhaus, H. Douglas Morris, Marjan Huizing, Shelley Hoogstraten-Miller (Invalid date). Retro-orbital injections in mice. LabAnimal.
10.1038/laban0511-155
LINK

29G x 12.7mm 0.3ml syringeUlticareCatalog #09230

Remove the needle slowly and press the eyelid shut for a few seconds.
Validating the injection was successful is possible with JF525 by looking at the pee of the animal after it wakes up.
Leaving the animal in a clean cup with kim-wips will collect the pee and it would seem pink.