May 30, 2025
DIO-SPOTlight mice tissue processing
- Matt Oliver1
- 1Duke University
Protocol Citation: Matt Oliver 2025. DIO-SPOTlight mice tissue processing. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpqno1lzp/v1
Manuscript citation:
Matthew L Oliver, Zachary F Caffall, Callie B Eatman, Timothy D Faw, Nicole Calakos
(2025) DIO-SPOTlight Transgenic Mouse to Functionally Monitor Protein Synthesis Regulated by the Integrated Stress ResponseeLife14:RP104457https://doi.org/10.7554/eLife.104457.1
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: May 29, 2025
Last Modified: May 30, 2025
Protocol Integer ID: 219161
Keywords: ASAPCRN, Perfusion , Tissue processing, spotlight mice tissue, perfusing mice, mice tissue, tissue slicing, tissue, cryostat, tissue processing, mice
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020607
Abstract
This protocol contains a relatively standard description of transcardially perfusing mice followed by tissue slicing using a cryostat.
Troubleshooting
DIO-SPOTlight mice tissue processing
Anesthetize mice using the drop jar method containing 3-5% aerosolized isoflurane.
Perform a toe pinch to ensure the mouse is fully anesthetized prior to pinning the limbs down.
Use scissors to open the thoracic cavity and perform a transcardial perfusion by inserting a needle in to the right ventricle and cut the top of the left lobe, immediately perfuse using ~25 ml ice cold PBS followed by ~30 ml ice cold 4% paraformaldehyde (PFA).
Cut off the head and remove the skin behind the ears to behind the eyes.
Open the skull and remove the brain.
Place the brain in 4% PFA + 20% sucrose in 1x PBS for 24 hours at 4C.
The following day transfer the brains to 30% sucrose in 1x PBS for an additional 24 hours at 4C.
Embed the tissue in optimal cutting temperature compound (OCT) and freeze.
The OCT embedded brains are then sliced cryostat into 40 um sagittal sections and store in 1x PBS with 0.01% sodium azide at 4C until IHC is performed.
If spinal cord dissections are performed following the removal of the brain use the following landmarks to dissect the spine: cervical, base of skull to caudal T1 lamina; thoracic, 0.6 cm segment centered on T9 lamina; and lumbar, identified based on L1-L6 nerve roots.
Repeat steps 6 - 9 with the exception that the spinal cord sections were directly mounted onto microscope sildes.
Acknowledgements
Spinal cord dissections performed by Timothy Faw.