A protocol to visualize on-target specific drug binding in mammalian tissue with cellular resolution using tissue clearing and click chemistry

Summary Here, we provide a protocol to visualize on-target specific drug binding in mammalian tissue with cellular resolution. By combining tissue clearing and click chemistry, this protocol allows fluorescence tagging of covalent drug binding in situ. In addition, the protocol is compatible with molecular marker staining for cell type identifications. For complete details on the use and execution of this protocol, please refer to Pang et al. (2022).


SUMMARY
Here, we provide a protocol to visualize on-target specific drug binding in mammalian tissue with cellular resolution. By combining tissue clearing and click chemistry, this protocol allows fluorescence tagging of covalent drug binding in situ. In addition, the protocol is compatible with molecular marker staining for cell type identifications. For complete details on the use and execution of this protocol, please refer to Pang et al. (2022).

BEFORE YOU BEGIN
Understanding drug actions in vivo is critical for developing effective therapies. Despite remarkable methodological advancement has been made to profile the drug-target interactions at the molecular level, a detailed understanding at the cellular level has not been established. Conventional strategies studying drug tissue distribution typically involves homogenizing the tissue/organ of interests, during which the spatial or cellular information is lost. Position emission tomography (PET) is widely utilized to study spatial drug distribution but lacks the resolution to resolve drug binding at cellular level. Given the high degree of cell type heterogeneity of mammalian tissue, especially in the central nervous system (CNS), it is desirable to visualize drug binding with cellular resolution, while maintaining compatibility with molecular characterizations.
To profile drug binding targets, drugs can be delicately modified with an alkyne handle. With copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) click reaction, a tag (such as biotin or fluorophore) can be introduced for proteomic scale analysis. Such a strategy has proven highly versatile in chemoproteomics studies (Parker and Pratt, 2020). However, direct click labeling in mammalian tissue for in situ drug mapping has been challenging due to potential side reaction and low signal to noise ratio (SNR). Herein, by integrating tissue clearing and click chemistry drug labeling, we addressed these challenges with CATCH, a newly developed strategy to visualize on target specific covalent drug binding with high resolution.
The synthesis and characterization of these probes, as well as any new probes the users would like to use, should be separately carried out with chemoproteomics studies. The protocol here only focuses on the histological and imaging applications of existing, pre-validated probes.

Institutional permissions
All experimental protocols were approved by the Scripps Research Institute Institutional Animal Care and Use Committee and were in accordance with the guidelines from the National Institute of Health.

Preparation of tilted tube rack
Timing: 10 min 1. Use two 2 mL Eppendorf tubes, remove the cap. 2. Attach the tubes to an autoclavable 4-way test tube rack with tapes ( Figure 1). This rack is specifically made for click reaction incubation and reaction. As the rack is tilted, it allows maximal agitation in a small reaction volume.

STEP-BY-STEP METHOD DETAILS
Preparation of mouse brain sample

Timing: 2 days
In this step, we will administer PF7845-yne and prepare brain samples. Please note that the transcardial perfusion is the preferred method for preparing conventional brain histology samples. Any conventional protocols for brain perfusion are compatible with CATCH (Gage et al., 2012). For nonbrain tissues, please follow standard histology preparation protocols for fixation and tissue dissection.
1. Intraperitoneal (i.p.) administration of 1 mg/kg PF7845-yne in a vehicle of 10% DMSO, 2% Tween-80 in saline with an insulin syringe. c. Carefully open chest to expose heart. d. Make a small incision at the right atrium. A small amount of dark venous blood should come out. e. Insert the needle into the left ventricle, perfuse in ice cold PBS (10 mL/min) to remove blood. f. Stop perfusion when the liver is without blood stain and the liquid flowing out is clear. It takes $ 2 min ($ 20 mL PBS) to fully remove blood.

Click incubation buffer
Note: Effective removal of blood can reduce side reaction in final click labeling (Figure 2, troubleshooting 1). For additional resources on mouse perfusion, please refer to (Gage et al., 2012;Wu et al., 2021).
Note: Signs of body twitching, tail flicking and head moving are signs of good PFA perfusion.
5. Decapitate the mouse with tough cut surgical scissors. 6. Dissect out brains, fix samples in 4% PFA perfusion fixative reagent, 4 C, overnight. a. Cut skin along the midline. Pull skin to the side to fully expose the skull. b. Make two lateral cuts underneath the brainstem. c. Cut skull along the midline over the cerebellum. d. Insert scissors near the eyes and sever the skull. e. Cut skull along the midline over the cortex to fully expose the brain. f. Use thumb and index finger to pull skull from the brain. Carefully peel off skull with forceps. g. Remove the brain from the skull for PFA fixation.
Note: For additional resources for brain dissection, please refer to (Gage et al., 2012;Wu et al., 2021).
Pause point: Uncleared PFA fixed samples can be stored in 4 C for 1-2 months. If storage buffer gets cloudy, samples should be discarded.

Timing: 3 days
In this step, we would perform tissue clearing with CLARITY, a hydrogel-based tissue clearing technique (Chung et al., 2013). CLARITY can remove lipid and render tissue transparent while preserving tissue architecture. Tissue clearing is critical to enable click reaction drug visualization in tissue (Pang et al., 2022). The steps below are adopted from a published CLARITY protocol (Tomer et al., 2014). Please refer to the original protocol for additional details.
11. Prepare A1P4 CLARITY solution on ice. Components for A1P4 CLARITY solution should be prechilled in 4 C prior to use. a. Weigh out required solid VA-044 in a tube and keep on ice. b. Sequentially add water, 103 PBS, 32% PFA, 2% bis-acrylamide, 40% acrylamide solution. c. Dissolve VA-044 initiator with ice cold water. d. Add dissolved VA-044 and mix solution by shaking. 12. Transfer tissue sections to A1P4 CLARITY solution. Solution should be filled close to top to minimize room for air.
Note: For $10 brain sections, use a screw top 5 mL Eppendorf tube. For >20 brain sections, we recommend using a 15 mL centrifuge tube.
13. Incubate sections in A1P4 CLARITY solution, overnight in 4 C with gentle shaking (80 RPM). 14. Connect vacuum desiccator to the vacuum pump and a nitrogen source (i.e., nitrogen tank). 15. Keep tube caps loose on top to facilitate gas exchange. Place tubes in the vacuum desiccator, RT (Figure 3). 16. Switch on pump to remove air, 1 min. 17. Flush in nitrogen from a nitrogen tank or any nitrogen source till the desiccator is filled with nitrogen. 18. Repeat steps 16 and 17 twice to ensure oxygen is fully removed. 19. Close nitrogen tank. Place samples under vacuum for 15 min at RT. 20. Flush in nitrogen. Open the chamber just enough to reach the tubes. With nitrogen flushing, close caps to prevent oxygen entrance.
CRITICAL: CLARITY involves a free radical polymerization process and oxygen will inhibit CLARITY polymerization. Ensure oxygen is removed as much as possible. Note: We have tested clearing temperature at 37 C-40 C, SDS concentration of 4%-8% and obtained similar labeling efficiency. Higher temperature and prolonged clearing time may cause protein loss and thereby not ideal for thin tissue sections (troubleshooting 2). For additional resources on CLARITY clearing steps, please refer to (Tomer et al., 2014).
Pause point: Cleared CLARITY samples can be stored in 4 C for up to a year without significant difference for labeling efficiency. However, we do recommend refreshing PBS-NaN 3 storage buffer every 3-4 months to prevent microbial growth.  In this step, we would perform click reaction in CLARITY cleared tissue to label drug in situ with an Alexa647 fluorescence dye. After click labeling, tissue can undergo secondary staining for molecular target identifications.

Prepare click incubation buffer.
a. Sequentially add PBS, DMSO, 1.25 mM AF647 picolyl azide. Gently pipette to ensure proper mixing. b. Pre-mix BTTP and CuSO 4 stock solution, the solution should turn light blue. c. Add Cu-BTTP pre-mixed solution. Gently pipette to ensure proper mixing. d. Aliquot click incubation buffer into 2 mL Eppendorf centrifuge tubes (200 mL/tube).
Note: We recommend preparing a master mix with 5% extra volume. For each coronal/ sagittal brain section, use 200 mL for incubation. Cu 2+ concentration can be adjusted between 50-150 mM (Cu to BTTP ratio of 1:2). AF647 picolyl azide has a Cu chelating group and has shown superior reaction kinetics compared to conventional azide tag (Uttamapinant et al., 2012).
28. Transfer cleared tissue to click incubation buffer, 1 section/tube. 29. Place tubes on the tilted tube rack (Figure 1), overnight incubation with gentle shaking (80 RPM), RT. The rack should be shed from light.
CRITICAL: Click reaction requires Cu(I) as catalyst. Without reducing agent sodium ascorbate, the incubation step allows Cu 2+ to diffuse evenly into tissue before reaction. It ensures labeling happens homogeneously across the whole tissue z axis (troubleshooting 3). We do not recommend accommodating multiple tissue sections in the same tube (troubleshooting 4).
30. Prepare click reaction buffer without sodium ascorbate as in step 27. 31. Aliquot into 2 mL Eppendorf centrifuge tubes (195 mL/tube). 32. Transfer tissue sections to click reaction buffer without sodium ascorbate, 1 section/tube. 33. Prepare fresh 100 mM sodium ascorbate solution. 34. Add 5 mL sodium ascorbate solution to each tube (200 mL in total). Gently pipette mix to initiate click reaction (troubleshooting 5). 35. Place tubes on the tilted tube rack (Figure 1) Pause point: CLARITY-based slides can be stored in the dark at RT for at least 1 week. For longer storage, we recommend storing them at the 4 C.

EXPECTED OUTCOMES
For PF7845-yne, fluorescent drug signal can be observed throughout the cortical region, thalamus, amygdala, with the highest abundance in the hippocampus ( Figure 5). As FAAH is a membrane protein (Egertová et al., 2003), drug binding would appear as membrane like structure when examined at sufficient resolution (for example, in Figure 6, at 2.49 micron/pixel in-plane resolution).

LIMITATIONS
CATCH allows for high resolution covalent drug binding mapping in intact tissue. CATCH is highly specific and maps drug binding across different brain regions and cell types. However, as reversible drug-target engagement will be lost during sample preparation, further efforts are still needed to retain reversible drug binding in situ. Meanwhile, the protocol is focused on drug imaging in 100-micron brain sections. Further scaling up imaging volume to whole organ, or even whole body, would require optimization in both click reaction and tissue clearing.   Non-specific click labeling shows blood vessel like structure (arrow sticks) in vehicle controls Images represent primary somatosensory cortex (S1). Scale bar, 20 mm.

Potential solution
Blood is not fully removed during perfusion. Make sure needle is properly positioned in the left ventricle. Extend PBS perfusion if necessary.

Problem 2
Tissue deforms during CLARITY clearing.
Potential solution CLARITY hydrogel is not well formed to protect tissue structure integrity. Make sure the degassing chamber is properly sealed. Meanwhile, oxygen is inhibiting polymerization, therefore A1P4 solution should fill the tube as much as possible. If vacuum set up is not readily available, consider other tissue clearing techniques including SHIELD, FDISCO, IDISCO and CUBIC3.0, all of which are also compatible with CATCH.

Problem 3
Click labeling is not homogeneous across the Z axis, with surface only labeling (Figure 8).

Potential solution
Refresh CuSO 4 stock with clean dH 2 O. Perform click incubation at 37 C with agitation. During reaction, increase reducing agent concentration up to 25 mM will further help click reaction penetration.

Problem 4
Click labeling is not homogeneous on the X-Y plane, with certain parts being dark (Figure 9).

Potential solution
Tissue is not sufficiently covered by buffer. During both incubation and reaction, the user should make sure all tissue samples are fully submerged in the tube. Ensure sufficient agitation during reaction incubation. Increase incubation temperature to 37 C if necessary. Problem 5 Click reaction labeling shows low signal intensity due to low target abundance.

Potential solution
Optimize microscope laser and acquisition settings. In click incubation and reaction (steps 27-34), increase Alexa-647 picolyl azide concentration up to 20 mM. Increasing CuSO 4 concentration to 300 mM (Cu to BTTP ratio of 1:2) will further increase labeling intensity.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Li Ye (liye@scripps.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate original dataset or code. ACKNOWLEDGMENTS Z.P. is funded by the Dorris Scholar Award. L.Y. is funded by the National Institutes of Health Director's New Innovator Award (DP2DK128800), NIDDK (DK114165 and DK124731), the Dana Foundation, the Whitehall Foundation, and the Baxter Foundation. We thank Benjamin Cravatt, Michael Schafroth, and Daisuke Ogasawara for their contribution to the work.

AUTHOR CONTRIBUTIONS
Z.P. wrote the protocols and prepared figures. L.Y. supervised the project. Z.P. and L.Y. reviewed and edited the manuscript.

DECLARATION OF INTERESTS
The design, step, and applications of the protocol are covered in a pending patent application from The Scripps Research Institute.