Apr 02, 2025
Photobleaching fluorescent signals from mouse brain sections for Projection-TAGs
- 1Washington University in St. Louis
- Projection-TAGs
Protocol Citation: Hannah Hahm, Lite Yang, Vijay Samineni 2025. Photobleaching fluorescent signals from mouse brain sections for Projection-TAGs. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4z748vo5/v1
Manuscript citation:
Yang L, Liu F, Hahm H, Okuda T… MR, Samineni VK. Projection-TAGs enable multiplex projection tracing and multi-modal profiling of projection neurons. bioRxiv [Preprint]. 2024 Apr 28:2024.04.24.590975.
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: April 02, 2025
Last Modified: April 02, 2025
Protocol Integer ID: 126041
Keywords: Photobleaching, Molecular profiling, Projection-TAGs
Funders Acknowledgements:
National Institute of Diabetes and Digestive and Kidney Diseases
Grant ID: 5R01DK128475-04
Abstract
This protocol describes the process of photobleaching Sun1-GFP signal from mouse brain sections for Projection-TAG experiments.
For more Projection-TAGs protocols, please visit: https://www.protocols.io/workspaces/projection-tags
Materials
Key equipment and reagents:
UV light (27 total watts, 9 LEDs * 3 watts, Manufacturer: OPPSK, Part Number: OK-047-C9BLUV, Item model number: C9BL, Amazon Standard Identification Number: B07RPV6852)
H2O2 (Sigma-aldrich, H1009-100ML)
NaOH (Sigma-aldrich, 72068-100ML)
Before start
1. Photobleaching effect may vary based on factors such as tissue section thickness, sample preparation process, and tissue type. Optimal photobleaching time should be determined with a pilot experiment.
2. Tissue sections should be sliced on a cryostat and stored in 1 x DEPC-PBS for no longer than 48 hours prior to photobleaching.
3. Set up the photobleaching paradigm in a dark room:
Elevate the UV light above a flat surface using two cardboard freezer boxes on each side. The distance between UV light bulb and the flat surface should be around 4’’.
4. Prepare the following solutions:
a. 1X DEPC-PBS
b. 5X Photobleach Solution
| A | B | C | D | E | |
| Stock conc. | Final conc. | 5X conc. | 10 ml for 5 wells | ||
| NaOH | 10 M | 24 mM | 120 mM | 120 ul | |
| H2O2 | 30% | 4.5% | 22.5% | 7.5 ml | |
| PBS | 1X | 1X | 1X | 2.4 ml |
Photobleaching
Photobleaching
Calculate the wells to be used for photobleaching. 8-10 brain sections can be photobleached in each well.
Add 8 ml of DEPC-PBS to each well in a 6-well plate.
Using a paint brush, transfer brain sections to a Netwell insert in the well containing DEPC-PBS.
Once all transfers are done, add 2 ml of 5X Photobleach Solution to each well.
Gently shake the plate to mix solution.
Immediately place the plate below the UV light. The wells containing brain sections should align with the UV light.
Note: photobleach solution breaks down tissue sections, it’s critical to minimize the time for the sections in contact with the solution.
Turn on the UV light and start photobleaching for 30 min.
Immediately transfer Netwell insert containing the brain sections to a petri-dish/well filled with DEPC-PBS.
Rinse sections in DEPC-PBS to remove residual Photobleach Solution and bubbles.
Mount the sections on a glass slide.
Dry the slides in a dark drawer for about 15 minutes.
Examine the photobleaching effect under microscope.
Note: Brain sections from the same animal without photobleaching and brain sections from animals without Projection-TAG injections should be prepared as controls.
Store the slides at -20C for two hours before moving to -80C for long-term storage.