Tissue clearing & immunostaining

Exceptional Protection of Fluorescent Proteins (FP) and Endogenous Molecular Information



Get the first step of tissue processing right with our easy & reliable solution kit

Superior Tissue Preservation

Versatile: Safeguards endogenous fluorescence, protein antigenicity, nucleic acids, and overall tissue architecture. Synergizes with a range of existing tissue processing approaches including SWITCH (Cell, 2015) and MAP (NBT, 2016). 

Easy, Fast & Reliable

Easy & Reliable: Superior tissue preservation vs. CLARITY, without the variability of hydrogel embedding. Compatible with both non-fixed and PFA-fixed tissues.

Fast: Streamlined protocol preserves samples in a matter of days with minimal steps.

Protects Tissue for Multi-Round Processing

Repeated Staining & Imaging: Protects tissue during FP-imaging and antibody labeling across individual rounds.


Standard Protocol:

Stopping Points: The protocol can only be stopped at the red circle points in the timeline above, so plan ahead! 

1. Transcardially perfuse the animal with ice-cold PBS. For mice, use about 20 mL and a 5 mL/min flow rate. For rats, use 200 mL and a 60 mL/min flow rate. We recommend using heparinized PBS to remove as much blood as possible (20 U/mL concentration). Make sure the fluid is running completely clear before next perfusing with ice-cold 4% PFA in PBS. Use the same amounts and flow rates as before. Be careful not to introduce air bubbles inside tubing. When the fluid comes out of the mouth or a lung swells, adjust the position of the needle in the heart. 

2. Dissect out the brain / organ of interest. Careful dissection is essential to preserve the sample’s structural integrity. 

3. Incubate the sample in 4% PFA in PBS for 24 hours at 4°C with shaking. If you are not ready to continue to the next step, The sample can be stored in 1X PBS with 0.02% sodium azide at 4°C until you are ready to continue. 

4. Before proceeding, please check the Expiration Date on the SHIELD-Epoxy bottle. If the solution is used after the expiration date the mechanical stability of the sample can be compromised. 

5. Prepare fresh SHIELD OFF Solution. Mix the following in the order shown in the table and keep on ice or move to 4°C after mixing. After adding each reagent, please vortex or mix well to prevent precipitate formation.

6. Incubate the sample in SHIELD OFF Solution at 4°C with shaking for 3 days for mouse brains or similar sized samples. Rat brains should be incubated for 6 days. If your sample’s smallest dimension is 1.5 mm or smaller, please stop here after incubation and continue to the Small Sample SHIELD-ON protocol. 

7. Pre-warm SHIELD ON Buffer to 37°C. For mouse brains or similar sized samples use 20 mL. Rat brains require 40 mL. 

8. Transfer the sample to SHIELD ON Buffer (RT) and incubate at 37°C with shaking for 24 hours. SHIELD preservation is now complete. The sample can be stored in 1X PBS with 0.02% sodium azide at 4°C for several months without significant loss of fluorescence signal and structural integrity. 

9. You may now proceed to the tissue clearing section of the protocol.


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