SHIELD 2.0 Protocol

Reagents / Equipment Required

This protocol is optimized to prepare mouse brains for subsequent mRNA labeling.

It is very important to complete the fixation steps quickly to minimize mRNA degradation.

Due to the timing of the protocols, it is best to perfuse on Wednesdays to prevent weekend steps.

1X PBS in ice: For each mouse, prepare 50 mL of 1X PBS with RNAse free water and store in ice for at least 30 minutes before perfusion.
SHIELD 2.0 Perfusion Solution: Prepare 60 mL for each mouse, split into 40 mL and 20 mL volumes. Store in ice for at least 30 minutes before perfusion. To make 60 mL, mix the following:
- 7.5 mL of 32% Paraformaldehyde solution
- 15 mL of SHIELD 2.0 4X Perfusion Buffer Solution (final concentration of 1X SHIELD 2.0 Perfusion Buffer Solution)
- 37.5 mL RNAse free DI water

Note: It is a good idea to prepare extra solution for contingency purposes.

Transcardially perfuse the mouse with 50 mL of ice-cold 1X PBS to drain the blood, followed by 40 mL of ice-cold SHIELD 2.0 Perfusion Solution.
Carefully extract the brain.
Incubate the brain in 20 mL of ice-cold SHIELD 2.0 Perfusion Solution for 24 hours at 4°C with orbital shaking.
Prepare 100 mL fresh ice-cold 1X PBSN (add 0.02% sodium azide to 1X PBS and mix) per sample.
Rinse the brain with 50 mL of ice-cold PBSN, pour out the solution, and add fresh ice-cold PBSN. Incubate overnight at 4°C with orbital shaking to wash.

SHIELD 2.0 Fixation Solution: Prepare 20 mL in a conical tube. Store on ice for at least 30 minutes before use. Mix the following:
- 5 mL SHIELD 2.0 4X Epoxy Solution (Thaw at RT and use the entire vial)
- 5 mL SHIELD 2.0 4X Perfusion Buffer Solution
- 10 mL RNAse free DI water

Perfuse and PFA fix the brain as above.
Incubate the whole mouse brain in 20 mL of SHIELD 2.0 Fixation solution at 4°C for 3 days with shaking.
After 3 days, move the sample to Room Temperature and incubate for 24 hours with shaking.
Prepare 100 mL fresh ice-cold 1X PBSN (add 0.02% sodium azide to 1X PBS and mix) per sample.
Rinse the brain with 50 mL of ice-cold PBSN, pour out the solution, and add fresh ice-cold PBSN. Incubate overnight at 4°C with orbital shaking to wash.

Section the tissue into 1 mm thick slabs.
Move the sections to Prototype Delipidation Buffer v2A and incubate for 24 hours at 37C with shaking.
Wash overnight in PBSN at RT with shaking.

Label the tissue with your protocol of choice.

Incubate the samples in 50% Prototype EasyIndex, 50% 2X SSCT at RT for 2-3 hours.
Move the samples to 100% Prototype EasyIndex and incubate at RT for 2-3 hours more or until transparent.
Mount and image!

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