Feb 22, 2024

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

DOI
dx.doi.org/10.17504/protocols.io.yxmvm7rznv3p/v2
  • 1HHMI Janelia Research Campus
Boaz Mohar
Open access
DOI: 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 22, 2024
Last Modified: February 22, 2024
Protocol Integer ID: 95629
Funders Acknowledgement:
HHMI
Abstract
How to prepare JF dyes for retro orbital injection.
Materials
STEP MATERIALS
ReagentDimethyl sulfoxide (DMSO)Sigma AldrichCatalog #D2650
ReagentPluronic™ F-127 (20% Solution in DMSO)Sigma AldrichCatalog #P3000MP
ReagentPBSInvitrogen - Thermo Fisher
Reagent29G x 12.7mm 0.3ml syringeUlticareCatalog #09230
Protocol materials
ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
Step 2
ReagentPluronic™ F-127 (20% Solution in DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3000MP
Step 3
ReagentPBSInvitrogen - Thermo Fisher
Step 4
Reagent29G x 12.7mm 0.3ml syringeUlticareCatalog #09230
Step 5
Dye preperation
Dye preperation
Take an aliqote with Concentration100 Mass Percent of dye from Temperature-20 °C storage.


Add Amount20 µL DMSO and do ~10 up and down with the pipeter


ReagentDimethyl sulfoxide (DMSO)Sigma AldrichCatalog #D2650


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


ReagentPluronic™ F-127 (20% Solution in DMSO)Sigma AldrichCatalog #P3000MP


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

Pipetting
Drew up to 100ul without bubbles to a 29G syringe
Reagent29G x 12.7mm 0.3ml syringeUlticareCatalog #09230



Follow the Retro-orbital injection protocol
Protocol
Retro-orbital injection of virus or dye in mice
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.
Critical
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 (2011). Retro-orbital injections in mice. LabAnimal.
LINK
10.1038/laban0511-155

Reagent29G x 12.7mm 0.3ml syringeUlticareCatalog #09230

Critical
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.

Optional