Mar 10, 2025

Public workspaceDIO-SPOTlight in vivo ISR Activation with Tunicamycin v.250227

DOI
dx.doi.org/10.17504/protocols.io.eq2ly6oergx9/v1
  • Matt Oliver1,2
  • 1Calakos Lab;
  • 2Duke University
Matt Oliver
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly6oergx9/v1
Protocol Citation: Matt Oliver 2025. DIO-SPOTlight in vivo ISR Activation with Tunicamycin v.250227. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6oergx9/v1
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
Created: February 27, 2025
Last Modified: March 10, 2025
Protocol Integer ID: 123547
Keywords: ASAPCRN, DIO-SPOTlight ISR activation
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020607
Abstract
Abstract
This protocol contains the steps and dosages used for the acute in vivo integrated stress response activation experiment using tunicamycin treatment in young mice (P12-14). (Wang et al., 2015) indicates may not be as effective in older mice.
In vivo tunicamycin treatment
In vivo tunicamycin treatment
Prepare tunicamycin (Tocris) stock solution in dimethyl sulfoxide (DMSO) to a concentration of 5 mg/ml. Prepare fresh, when possible and discard after 1 month.
a. On the day of treatment, dilute tunicamycin 1:25 (v/v) in 150 mM dextrose in PBS with 5% Tween-80
b. Prepare vehicle treatment by 1:25 (v/v) dilution of DMSO in 150 mM dextrose in PBS with 5% Tween-80
i. Sterile filter the tunicamycin and vehicle solutions through separate 0.22 um syringe filters
Starting around 3:00 PM, weigh P12 pups and record weights
Prepare sterile syringes to administer tunicamycin or vehicle solutions at a final concentration of 3 ug/g bodyweight.
Scruff mouse pup and clean the injection site with an alcohol wipe.
Inject tunicamycin or vehicle solution subcutaneously at the base of the neck and return the mouse to the cage.
Perform a second identical injection 2 hours after the first.
Sacrifice mice 16 – 18 hours  later.
Protocol references
Wang, H., Wang, X., Ke, Z. J., Comer, A. L., Xu, M., Frank, J. A., Zhang, Z., Shi, X., & Luo, J. (2015). Tunicamycin-induced unfolded protein response in the developing mouse brain. Toxicology and Applied Pharmacology, 283(3), 157–167. https://doi.org/10.1016/j.taap.2014.12.019