Immunolabeling of a Whole Mouse Brain
Naveen Ouellette, Andrew Recknagel, Kevin Cao, Judith Baka, Molly Logsdon, Jayaram Chandrashekar
Abstract
Our mice have sparse viral labeling of target neuron populations with genetically encoded fluorescent proteins (FPs). Delipidation ensures that whole brain samples become optically clear and more accessible to antibody labeling. However, the signal from endogenous FPs is often dimmed or quenched during organic solvent delipidation steps. Tissue expansion also dilutes the concentration of fluorophores. To amplify signals of FPs for high resolution, multi-scale imaging, we immunolabel delipidated brain samples.
Before start
If performing the Whole Mouse Brain Delipidation, Immunolabeling, and Expansion Microscopy protocol, start with a brain that has been delipidated using the Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain and Aqueous (SBiP) Delipidation of a Whole Mouse Brain protocols.
Steps
Immunolabeling of a Whole Mouse Brain
Start with a whole mouse brain perfused with 4% PFA, post-fixed, and stored in PBS in a 20 mL vial.
Using a 20 mL glass vial, wash the brain with PTxw, rotating at Room temperature
Transfer the brain to a small 4 mL vial containing diluted primary antibody in ~ 4mL PTxw and incubate with rocking, a nutator or shaker, at Room temperature for 11 days.
- 10 μg/brain for primary antibody
- 20 μg/brain for primary conjugated antibody
Transfer the brain to a 20 mL glass vial and wash with PTxw, rocking on a nutator or shaker at Room temperature, and replacing solution for each step:
- PTxw for
1h 0m 0s - PTxw for
2h 0m 0s - PTxw for
4h 0m 0s - PTxw
4h 0m 0s - PTxw for
24h 0m 0s - PTxw for
24h 0m 0s - PTxw for
24h 0m 0s
Transfer the brain to a small 4 mL vial containing diluted secondary antibody in ~4mL PTxw and incubate with rocking at Room temperature for 11 days.
- 20 μg/brain for secondary antibody
Transfer brain to a 20 mL glass vial and wash with PTxw, rocking at Room temperature, and replacing solution for each step:
- PTxw for
1h 0m 0s - PTxw for
2h 0m 0s - PTxw for
4h 0m 0s - PTxw
4h 0m 0s - PTxw for
24h 0m 0s - PTxw for
24h 0m 0s - PTxw for
24h 0m 0s
Wash the brain with 1X PBS, rocking at Room temperature .
