ARCHmap Video
ARCHmap Step-by-step Protocol
SUMMARY
ARCHmap (Active CleaRing with CUBIC-LH, Hydrogel-embedding, and RI-matching with PuClear) is an efficient aqueous-based protocol for homogeneous clearing of the entire mouse body. This protocol details a comprehensive workflow, including:
• Perfusion & fixation
• Decalcification
• Delipidation (CUBIC-LH-based)
• Whole-body immunofluorescence staining (optional)
• Hydrogel embedding
• Refractive index (RI) matching
Cleared samples are compatible with blockface-VISoR imaging, enabling high-resolution anatomical mapping of the peripheral nervous system and associated tissues throughout the entire mouse body.
PREPARATORY PROCEDURES & EQUIPMENT SETUP
1. Custom Perfusion Needle Preparation
1.
Trim the sharp tip of an intravenous infusion needle with a 0.6 mm outer diameter using wire cutters.
2.
Polish the cut surface with sandpaper to eliminate sharp edges.
3.
Cover the needle with a polytetrafluoroethylene (PTFE) tube extending ~1 mm beyond the needle tip.
CRITICAL: This PTFE sheath prevents cardiac tissue puncture from the sharp metal tip during perfusion and circulation.
2. Circulating Perfusion System Assembly
A custom-built closed-loop circulation system is used for fixation, decalcification, and delipidation. The system includes:
•
a custom infusion needle (insert into the left ventricle),
•
a plastic chamber with a lid for holding a mouse sample and perfusion reagents,
•
an outlet silicone tubing connecting a peristaltic pump to the infusion needle,
•
an inlet silicone tube connecting the plastic chamber to the peristaltic pump to maintain a closed circulation loop,
•
an outlet silicone tubing is connected using plastic hose barb union or Luer connectors to ensure secure, leak-free junctions,
•
a peristaltic pump for maintaining flow,
•
a 10-µm nylon syringe filter inserted the silicone tubing to prevent needle clogging during long-term perfusion.
ANIMAL PREPARATION
1. Food deprivation and gastrointestinal empty
🕐
Duration: 1 day
1.
Deprive food and water for 24 h, while providing 200 ml 7% w/v polyethylene glycol electrolyte solution in 5% sucrose to empty the gastrointestinal tract (Table 1).
| Reagent | Amount |
|---|---|
| Polyethylene glycol electrolytes | 14 g |
| Sucrose | 10 g |
| H₂O | To 200 ml |
2. Anesthesia and pre-perfusion preparation
🕐
Duration: 1 hour
2.
Prepare 300 ml of 4% paraformaldehyde (PFA) in 0.1 M PBS (Table 2), or use commercial 4% PFA (special for electron microscopy). This solution will be used for fixation and postfixation.
| Reagent | Amount |
|---|---|
| 8% Paraformaldehyde | 150 ml |
| 10× PBS | 30 ml |
| ddH₂O | 120 ml |
3.
Prepare 1% sodium pentobarbital in normal saline (Table 3).
| Reagent | Amount |
|---|---|
| Sodium pentobarbital | 0.1 g |
| Normal saline | 10 ml |
4.
Anesthetize mice by intraperitoneal injection of 1% sodium pentobarbital at a dosage of 100 mg/kg. The mouse is deeply anesthetized when both the tendon reflex and blink reflex disappeared.
5.
Shave the hair off using an electric clipper. Apply depilatory cream to remove any remaining hair. Wait for 10 minutes, then gently wipe off the softened hair and residual cream using tissue paper.
6.
Rinse thoroughly with warm water and dry the skin gently.
7.
Vasculature Labeling (optional).
a.
To label the vasculature throughout the body, pre-warmed DyLight 649-conjugated lectin at 37°C 10 minutes before use.
b.
Use an insulin syringe(31G × 8 mm) to slowly inject 0.2 ml of the pre-warmed lectin solution into the tail vein of the anesthetized mouse.
c.
Keep the mouse heart beating to allow lectin circulating systemically for 2 minutes, then immediately proceed with transcardial perfusion as described in the following section.
CRITICAL: After anesthesia and hair shaving, mice are prone to hypothermia. Use a heating pad to maintain body temperature during preparation.
STEP-BY-STEP PROTOCOL
1. Transcardial Perfusion & Postfixation
🕐
Duration: 1 day
8.
After hair shaving, position the mouse supine on a foam board and secure all four limbs using pins. All subsequent perfusion steps should be conducted inside a fume hood.
9.
Connect the perfusion system with a peristaltic pump, and flush with pre-warmed 0.1 M PBS at 37°C to eliminate any air bubbles in the tubes.
10.
Make a horizontal incision just above the diaphragm to expose the thoracic cavity using an ophthalmic scissor. Extend the cut upward along both sides of the rib cage to make a U-shaped flap. Secure the chest wall with a small hemostatic clip, and turn over the chest wall to fully expose the heart.
11.
Insert the custom infusion needle into the left ventricle of the heart, and secure it at the cardiac apex using a small hemostatic clip. Then, use an ophthalmic scissor to make a small incision in the right atrial appendage to allow for outflow of perfusate.
12.
Perfuse the mouse with 40 ml of normal saline at 37°C at a flow rate of 10 ml/min to flush the blood.
13.
Follow with 20 ml of 4% PFA in 0.1 M PBS at room temperature at the same flow rate (10 ml/min).
14.
Secure the limbs, tail, and infusion tubing to the foam board using nylon zip ties to stabilize body posture and to prevent the needle from dislodging.
15.
Immerse the entire mouse in a chamber filled with 250 ml of 4% PFA.
16.
Submerge the end of the inlet tube below the liquid surface to maintain a closed-loop circulation.
17.
Insert a 10-µm nylon syringe filter into the middle of the silicone tubing to prevent particles from clogging the needle.
18.
Remove air in the tubes, and circulate the fixative at a flow rate of 5 ml/min for 24 hours at room temperature in the dark.
CRITICAL: A well-circulated mouse sample should be bouffant after postfixation. If not, adjust the position of the perfusion needle, or discard this sample.
2. Decalcification
🕐
Duration: 4 days
19.
Prepare 600 ml of 11.5% w/v Ethylenediaminetetraacetic acid disodium salt (EDTA-Na₂) (pH 8.0) and 600 ml of 23% w/v EDTA-Na₂ (pH 8.0) for each mouse in advance.
20.
To prepare 1 L of EDTA-Na₂ solution, first dissolve 13.5 g and 25.5 g of NaOH pellets in 500 ml ddH₂O using a magnetic stirrer for 11.5% and 23% w/v EDTA-Na₂, respectively (Table 4 and 5). Once fully dissolved, slowly add 115 g and 230 g of EDTA-Na₂ for 11.5% and 23% w/v EDTA-Na₂, respectively (Table 4 and 5), while continuing stirring. Adjust the final volume to 1 L with ddH₂O and the pH to 8.0.
| Reagent | Amount |
|---|---|
| EDTA-Na₂ | 115 g |
| NaOH | 13.5 g |
| ddH₂O | To 1 L |
| Reagent | Amount |
|---|---|
| EDTA-Na₂ | 230 g |
| NaOH | 25.5 g |
| ddH₂O | To 1 L |
21.
Pause the peristaltic pump and discard the 4% PFA from the perfusion chamber. Intracardially rinse the mouse sample with 0.1 M PBS for 1 hour at a flow rate of 5 ml/min to remove residual PFA.
22.
Discard the PBS and add 300 ml of 11.5% EDTA-Na₂ in the chamber. Transfer the chamber to a 37°C water bath, and intracardially circulate with 11.5% EDTA-Na₂ solution for 2 days. Refresh the solution on the second day.
23.
Intracardially circulate with 300 ml of 23% EDTA-Na₂ (pH 8.0) for another 2 days. Similarly, refresh the solution on the second day.
CRITICAL: Replace the 10-µm nylon syringe filter daily during the entire circulation process to prevent filter clogging.
CRITICAL: After the first day of decalcification, check the decalcification effect by gently bending the paws of mouse. If no significant softening is observed in any one paw, adjust the position of the perfusion needle and extend the decalcification time accordingly.
24.
After decalcification, intracardially rinse the sample with 0.1 M PBS three times for three hours each to fully remove residual EDTA-Na₂.
3. Delipidation with CUBIC-LH
🕐
Duration: 7 days
25.
To prepare CUBIC-LH solution, add 100 g of Triton X-100 and 150 g of N-Butyldiethanolamine in 750 ml of ddH₂O when stirring at room temperature until completely dissolved (Table 6).
| Reagent | Amount |
|---|---|
| Triton X-100 | 100 g |
| N-Butyldiethanolamine | 150 g |
| ddH₂O | 750 ml |
26.
Replace the PBS in the chamber with 300 ml of CUBIC-LH clearing solution.
27.
Intracardially circulate the whole-body sample at 37°C, and maintain continuous circulation for 6 days. Refresh the clearing solution daily for the first 2 days, and every other day thereafter.
28.
After delipidation, intracardially rinse the sample with 0.1 M PBS at 37°C three times for 8 hours each to thoroughly remove residual clearing reagents.
CRITICAL: Ensure no air was perfused into the mouse sample during clearing and washing.
4. Whole-Body Immunostaining (optional)
🕐
Duration: 16 days
To perform immunostaining, we employed a dual-catheter perfusion strategy by inserting one needle into the left ventricle (as described above), and a second needle into the abdominal cavity of the cleared whole-body sample.
29.
Prepare the pretreating solution as in Table 7. Intracardially circulate the sample with 200 ml of pretreating solution at 37°C overnight to enhance tissue permeability prior to immunostaining. The pretreating solution needs to be freshly prepared before use.
| Reagent | Amount |
|---|---|
| Triton X-100 | 400 µl |
| DMSO | 40 ml |
| Glycine | 4.52 g |
| ProClean (or 5% Sodium azide) | 200 µl |
| 0.1 M PBS | To 200 ml |
30.
Prepare the blocking solution as in Table 8. Intracardially circulate the sample with 200 ml of blocking solution at 37°C for 1 day to minimize non-specific binding.
| Reagent | Amount |
|---|---|
| Triton X-100 | 400 µl |
| DMSO | 20 ml |
| Donkey serum | 6 ml |
| ProClean (or 5% Sodium azide) | 200 µl |
| 0.1 M PBS | To 200 ml |
31.
Prepare PTwH as in Table 9. Wash the sample three times with PTwH for 1 hour each at 37°C.
| Reagent | Amount |
|---|---|
| Tween-20 | 2 ml |
| Heparin | 10 g |
| 0.1 M PBS | 1 L |
32.
Intracardially circulate the sample with primary antibody.
a.
Prepare the antibody incubation solution freshly on the day of use as in Table 10.
| Reagent | Amount |
|---|---|
| Triton X-100 | 400 µl |
| DMSO | 10 ml |
| Donkey serum | 4 ml |
| ProClean (or 5% Sodium azide) | 200 µl |
| PTwH | To 200 ml |
b.
Incubate the sample with 200 ml of primary antibody solution at 37°C for 7 days. For example, to label sympathetic nerves, dilute 400 μl of rabbit anti-tyrosine hydroxylase (TH) antibody (Merck, AB152) in 200 ml of the antibody incubation solution (1:500 dilution).
c.
For the first 5 days, intracardially circulate the antibody solution via the ventricular intravenous infusion needle.
d.
Stop intracardial circulation of antibody on day 6. Insert a blunt-tipped needle into the abdominal cavity, and create a small puncture in the lower abdominal wall to improve antibody penetration into visceral tissues.
e.
Reconnect the outlet tubing to the needle inserted into the abdominal cavity.
f.
Start abdominal cavity circulation of the antibody solution for another 2 days at 37 °C. All circulation steps are performed at a constant flow rate of 5 ml/min, with a 10-µm syringe filter in-line to prevent clogging.
33.
Wash to remove unbound primary antibody.
a.
Reconnect the outlet silicone tubing to the ventricular infusion needle.
b.
Wash the sample three times with PTwH for 5 hours each at 37 °C.
c.
Reconnect the tubing to the abdominal catheter and perform two additional abdominal washes under the same conditions.
d.
The total washing time is approximately 1 day.
34.
Intracardially circulate the sample with secondary antibody.
a.
Prepare 200 ml of antibody incubation solution as in Table 10.
b.
Add 400 μl of donkey anti-rabbit Alexa Fluor 647 (or another appropriate secondary antibody) at a 1:500 dilution. Prepare the solution freshly and keep it from light.
c.
Circulate the sample at 37°C for 5 days in total. For the first 4 days, intracardially circulate the secondary antibody solution via the ventricular intravenous infusion needle.
d.
On day 5, switch to abdominal cavity perfusion. Disconnect the silicone tubing from the Luer connector on the heart catheter, and reconnect it to the abdominal needle. Continue abdominal circulate of the secondary antibody solution for another day at 37°C.
35.
Wash to remove unbound secondary antibody.
a.
Reconnect the tube to the ventricular infusion needle and wash the sample three times with PTwH for 5 hours each at 37 °C.
b.
Reconnect the tubing to the abdominal needle and perform two additional abdominal washes under the same conditions.
5. Hydrogel Embedding
🕐
Duration: 2 days
36.
Prepare the 8% HMS-iohexol solution and 50% (w/v) bovine serum albumin (BSA) solution one day in advance (Table 11 and Table 12). 8% HMS-iohexol solution and 50% (w/v) BSA solution should be prepared with gently shaking (60 rpm) at 4°C and room temperature, respectively, to ensure complete dissolution.
| Reagent | Amount |
|---|---|
| Iohexol | 67 g |
| 8% Paraformaldehyde | 50 ml |
| Acrylamide | 8 g |
| N,N′-Methylenebisacrylamide | 0.1 g |
| 10× PBS | 10 ml |
| Reagent | Amount |
|---|---|
| BSA | 50 g |
| ddH₂O | 60 ml |
CRITICAL: Use freshly purchased, high-quality BSA for preparing the 50% (w/v) solution. Aged or degraded BSA may form insoluble clumps during mixing. Quickly manually shake the mixture just after adding powder into water will accelerate dissolution of BSA.
37.
After fully dissolved, transfer the BSA solution to a 4 °C refrigerator for defoaming and cooling. To prepare the embedding solution, mix equal volumes of 8% HMS-iohexol solution and 50% (w/v) BSA solution to create the HMS and BSA mixture.
CRITICAL: The BSA solution should be cooled to 4 °C, or it will lead to premature polymerization after mixing. The HMS-iohexol solution should be slowly added into the BSA solution with hand stirring to prevent formation of insoluble clumps. Store the final embedding solution at 4 °C for defoaming prior to sample immersion.
38.
Add 0.5 g of VA-044 thermal initiator to 200 ml final embedding solution immediately before use, and mix thoroughly on ice. Immerse each whole-body sample with 200 ml of this solution at 4 °C on a shaker at 60 rpm for 2 days for complete tissue infiltration.
CRITICAL: Add the polymerization initiator (VA-044) just before immersion, or it may trigger early polymerization even at low temperature.
39.
On each day of immersion, use a 5 ml syringe to carefully inject 5-10 ml of the freshly prepared embedding solution into the abdominal and cranial cavity of the cleared mouse to improve internal diffusion. Take care to avoid introducing air bubbles during injection, as trapped air may compromise subsequent imaging quality.
CRITICAL: Avoid introducing air bubbles during injection as they may compromise imaging quality.
40.
Transfer the sample into a 3D-printed mold coated with hydrophobic material after two days' immersion.
41.
Use the tail of the mouse sample to hold onto the mold to prevent the sample from floating up.
42.
Add 100 ml of fresh embedding solution to the mold to fully immerse the sample.
43.
Place the sample into a plastic container on ice.
44.
Transfer the container together with the sample into a vacuum desiccator.
45.
Evacuate the chamber for 20 minutes to fully remove dissolved and ambient air. Then, close the valve between the vacuum pump and the desiccator, and shut off the vacuum pump.
46.
Introduce nitrogen gas through the valve between the desiccator and gas cylinder to restore atmospheric pressure in an oxygen-free environment. Once pressure is equilibrated, take out the sample, and remove foam on the surface of embedding solution. Remove ice and add approximately 10 ml of water in the bottom of the container to maintain humidity. Then, seal the plastic container immediately.
CRITICAL: Do not add full volume of embedding solution in the mold. Excessive bubble formation may cause the solution to overflow from the mold during vacuum degassing, because the embedding solution is highly viscous. If this occurs, pause the vacuum process and enable bubbles to dissipate naturally before resuming evacuation.
47.
Transfer the sealed container with the sample into a 37 °C incubator, and polymerize for 2.5 hours.
48.
After polymerization, carefully remove the sample from the mold. Simply rinse the sample with clean water, and dry it using lint-free tissue paper.
6. Refractive index (RI) matching
🕐
Duration: ≥ 10 days
49.
Prepare the PuClear RI-matching solution 2 days in advance for complete dissolution. To prepare ~1 L of solution, first add ddH₂O and urea into a 1 L reagent bottle, followed by 2,2′,2″-Nitrilotriethanol and finally iohexol (Table 13). Seal the bottle tightly, and place it on a shaker at 200 rpm at 37°C overnight to ensure complete mixing. After dissolution, store the solution at room temperature. The final RI of the solution should be approximately 1.52.
| Reagent | Amount |
|---|---|
| Iohexol | 650 g |
| Urea | 300 g |
| 2,2′,2″-Nitrilotriethanol | 140 g |
| ddH₂O | 210 ml |
CRITICAL: Although RI matching may induce minor cracks within the embedding hydrogel, these structural imperfections do not compromise the integrity of whole-body imaging.
50.
Transfer the embedded whole-body sample into a plastic container, and fully immerse it in about 300 ml of PuClear RI-matching solution.
51.
Wrap the container with aluminum foil to protect the sample from light.
52.
Place the sealed container on a shaker at 60 rpm for 10 days to allow complete RI matching.
53.
Refresh the PuClear solution 3 days before imaging.
CRITICAL: Although RI matching may induce minor cracks within the embedding hydrogel, these structural imperfections do not compromise the integrity of whole-body imaging.
54.
Samples can be stored in PuClear RI-matching solution at room temperature in the dark for at least 2 months.
| Procedures | Reagents | Time |
|---|---|---|
| Perfusion and postfixation | 4% PFA | 1 day |
| Decalcification | 10% EDTA | 2a days |
| 20% EDTA | 1a day and 15 hours | |
| 0.1M PBS | 9 hours | |
| Delipidation | CUBIC-LH | 6b days |
| 0.1M PBS | 1 day | |
| Immunohistochemistry (optional) | Pretreating solution | 1 day |
| Blocking solution | 1 day | |
| Primary antibody | 7 days | |
| 0.1M PBS | 1 day | |
| Secondary antibody | 5 days | |
| 0.1M PBS | 1 day | |
| Embedding | 4% HMS-iohexol, 25% BSA | 2 days |
| RI-matching | PuClear | 10c days |
| Total time | 24 days (without immunostaining) / 40 days (with immunostaining) | |
aBuffer refreshed daily.
bBuffer refreshed daily for the first two days, and every other day afterwards.
cBuffer refreshed three days before imaging.
bBuffer refreshed daily for the first two days, and every other day afterwards.
cBuffer refreshed three days before imaging.