Materials and Reagents

C57BL/6NCrSlc mice (6–8-week-old male; Japan SLC)
50 mL polypropylene conical tube (Corning, Falcon^®, catalog number: 352070)
15 mL polypropylene conical tube (Corning, Falcon^®, catalog number: 352096)
1 mL syringe for tuberculin slip tip (TERUMO, catalog number: SS-01T)
Injection needle 25G × 1 (TERUMO, catalog number: NN-2525R)
Insulin syringe (with 27G × 1/2 needle) (TERUMO, catalog number: SS-10M2713)
Normal winged needle for vein D type (25G × 5/8 needle) (TERUMO, catalog number: SV-25DLK)
Three-way stopcock (TERUMO, catalog number: TS-TL1K)
30 mL syringe lock type 30 (TERUMO, catalog number: SS-30LZ)
5 mL Costar Stripette (Corning, catalog number: 4487)
10 mL Costar Stripette (Corning, catalog number: 4488)
25 mL Costar Stripette (Corning, catalog number: 4489)
MOG_35-55 (MEVGWYRSPFSRVVHLYRNGK, Sigma-Aldrich)
Incomplete Freunds’ adjuvant (IFA) (Sigma-Aldrich, catalog number: F5506)
Mycobacterium tuberculosis H37RA (DIFCO, catalog number: 231141)
PTx from Bordetella pertussis (Sigma-Aldrich, catalog number: P7208-50UG)
Saline (Otsuka Pharmaceutical, catalog number: K6E85)
Isoflurane (Pfizer)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625)
Potassium chloride (KCl) (Kanto Chemical, catalog number: 32326-00)
Disodium hydrogen phosphate (Na₂HPO₄) (Nacalai Tesque, catalog number: 31801-05)
Potassium dihydrogen phosphate (KH₂PO₄) (Kishida Chemical, catalog number: 000-63955)
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034)
Sodium hydroxide (NaOH) (FUJIFILM Wako Chemicals, catalog number: 198-13765)
4% paraformaldehyde phosphate buffer solution (PFA) (Nacalai Tesque, catalog number: 09154-85)
Triton X-100 (Sigma-Aldrich, catalog number: T8787)
Sodium azide (FUJIFILM Wako Chemicals, catalog number: 197-11091)
Casein from bovine milk (Sigma-Aldrich, catalog number: C3400)
1,2-Hexanediol (Tokyo Chemical Industry, catalog number: H0688)
1-Methylimidazole (Tokyo Chemical Industry, catalog number: M0508)
2,3-Dimethyl-1-phenyl-5-pyrazolone (Tokyo Chemical Industry, catalog number: D1876)
Nicotinamide (Tokyo Chemical Industry, catalog number: N0078)
Silicone oil (Shin-Etsu Silicone, catalog number: HIVAC-F4)
Mineral oil (Sigma-Aldrich, catalog number: M8410-1L)
Anti-actin, α-smooth muscle (α-SMA)–FITC antibody, mouse monoclonal (clone: 1A4) (Sigma-Aldrich, catalog number: F3777)
Purified rat anti-mouse CD31 (clone: MEC 13.3) (BD Biosciences, catalog number: 550274)
Goat anti-rat IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor^TM 555 (Thermo Fisher Scientific, catalog number: A-21434)
Dimethyl sulfoxide (DMSO) for spectrochemical analysis (FUJIFILM Wako Chemicals, catalog number: 045-28335)
Sodium borohydride (NaBH₄) (Tokyo Chemical Industry, catalog number: S0480)
Ethanol (Kanto Chemical, catalog number: 14033-00)
[1,2-Bis(diphenylphosphino)ethane]dichlorodigold(I) (Au₂(dppe)Cl₂) (Sigma-Aldrich, catalog number: 717363)
Dichloromethane (Kanto Chemical, catalog number: 10158-81)
Hydrochloric acid (FUJIFILM Wako Chemicals, catalog number: 080-01061)
Acetone (Kanto Chemical, catalog number: 01026-81)
Methanol (Kanto Chemical, catalog number: 25183-81)
[Au₁₃(dppe)₅Cl₂]Cl₃ (Au₁₃) [synthesized according to the literature (Shichibu and Konishi, 2010), see Recipes]
1 M HEPES buffer, pH 7.2 (see Recipes)
Phosphate buffered saline (PBS) (see Recipes)
Delipidation/decoloration buffer (see Recipes)
Staining buffer (see Recipes)
CUBIC-R (see Recipes)
Observation oil (see Recipes)

Equipment

Micro-dissecting scissors (Hammacher, catalog number: HSB014-11)
Micro scissors 105 mm straight sword (AS ONE, catalog number: YS-7105)
Angled serrated tip forceps (Hammacher, catalog number: HSC187-11)
KFI tweezers GG (Kowa Forceps Industry, catalog number: K-3 GG)
PIPETMAN P (Gilson, model: P20, catalog number: F123600)
PIPETMAN P (Gilson, model: P200, catalog number: F123601)
PIPETMAN P (Gilson, model: P1000, catalog number: F123602)
Pipet-aid (FALCON/catalog number: 357590)
Light sheet microscopy (LaVision BioTec/UltraMicroscope II)
Shake-LR (TAITEC, catalog number: 0054809-000)
Wave-SI (TAITEC, catalog number: 0054334-000)
Double shaker NR-30 (TAITEC, catalog number: 0000205-000)
Neo shaker NS-LR (AS ONE, catalog number: 2-7827-01)
Stereomicroscope (OLYMPUS, model: SZ61)
LED illuminator stand (OLYMPUS, model: SZ2-ILST)
Refractive index measurement equipment (ATAGO, catalog number: PAL-RI)

Software

Imaris version 9.0.0 (Bitplane, https://imaris.oxinst.com)

Procedure

Figure 1 shows an overview of the protocol.

Figure 1. Overview of procedures for microinflammation detection using Au₁₃ nanoclusters and the CUBIC tissue-clearing method. Mice were immunized with MOG_35-55/CFA (IFA containing M. tuberculosis H37RA) and underwent PTx injection. On days 16–20, when experimental autoimmune encephalomyelitis (EAE) develops with clinical scores of 2–3, the EAE mice were injected with Au₁₃ nanoclusters, anesthetized after 2 h, and transcardially perfused with PBS and then PFA. The spine with the ribs was taken out and further subjected to PFA fixation for one day. Then, L1–L6 were excised carefully and immersed in delipidation/decoloration reagent (hexanediol/methylimidazole) for four days (days 1–5). After delipidation and decoloration, L1–L6 were stained with anti-SMA or anti-CD31 for seven days (days 5–12) and subjected to refractive index adjustment in CUBIC-R (days 12–14). The Au signal was visualized by light sheet microscopy and analyzed by Imaris software.

Induction of EAE
1. Mix 100 μL (400 μg) of 4 mg/mL of MOG_35-55in PBS and 100 μL of CFA [10 mL of IFA plus two vials (20 mg) of M. tuberculosis H37RA] in two syringes using a three-way stopcock. Emulsify by pressing the syringe back and forth, transferring the contents from one syringe to the other for 5–10 min until a stable white emulsion is produced (for details, see Tanaka et al., 2017).
2. Immunize C57BL/6 mice with 200 μL of MOG_35-55/CFA subcutaneously (s.c.) at the tail base using a 1 mL syringe with a 25G needle on day 0.
3. Inject i.v. 400 ng of PTx in 200 μL of saline into the tail vein of mice using an insulin syringe on days 0, 2, and 7.
4. Assess clinical scores of EAE daily with a 0–5 scoring system (0, no signs; 1, flaccid tail; 2, impaired righting reflex and/or gait; 2.5, paralysis of one hind limb; 3, paralysis of both hind limbs; 4, hind limb paralysis with partial fore limb paralysis; 5, moribund or dead).

Preparation of Au₁₃ nanoclusters
1. Synthesize Au₁₃ nanoclusters ([Au₁₃(dppe)₅Cl₂]Cl₃; see Recipe 1) and isolate as crystalline solids according to the procedures described in Shichibu and Konishi (2010).
2. Store 160 μM Au₁₃ nanoclusters in DMSO.

Fixation of spines from Au₁₃ nanoclusters–treated EAE mice
1. Prior to injecting into mice, Au₁₃ nanoclusters are diluted 5-fold with 10 mM HEPES (pH 7.2, see Recipe 2) or saline to 32 μM.
2. Inject i.v. 200 μL of 32 μM Au₁₃ nanoclusters into the tail vein of aEAE mice with clinical score 2–3 using the insulin syringe.
3. After 2 h, anesthetize the mice with 4% isoflurane.
4. Perfuse the mice with PBS (see Recipe 3) and PFA using the winged needle and three-way stopcock locked by syringes.
  1. Make an incision in the right atrial appendage of the heart.
  2. Perfuse with 25 mL of cold PBS via the left ventricle of the heart using the syringe with the 25G winged needle.
  3. After the PBS perfusion, change the solution to 4% PFA using the three-way stopcock.
5. Perfuse with 20 mL of cold 4% PFA.
6. Take out the spine with the ribs (Video 1) and immerse in 40 mL of 4% PFA in a 50 mL tube at 4 °C for 24 h with shaking at 60 rpm in an orbital motion on a TAITEC Shake-LR.
  
  Video 1. Procedures for the L1-L6 preparation. After transcardial perfusion with PBS followed by PFA, the spine with the ribs was immediately taken out and further fixed with PFA. Then, L1–L6 were excised by carefully cutting the intervertebral discs between T13-L1 and L6-S1 with a razor, with the T13 rib as a position marker under stereomicroscopy installed with an LED illuminator.

Delipidation/decoloration
1. Wash the spine with the ribs with 40 mL of PBS at room temperature for 30 min three times while shaking at 30 rpm in a seesaw motion on a TAITEC Wave-SI.
2. Excise L1–L6 carefully under stereomicroscopy installed with the LED illuminator (Video 1).
3. Immerse L1–L6 in 40 mL of delipidation/decoloration buffer (see Recipe 4) in a 50 mL tube at 37 °C for 4 days with shaking at 80 rpm in a reciprocating motion on an AS ONE Neo shaker NS-LR.

Antibody staining
1. Wash L1–L6 with 40 mL of PBS at room temperature for 1 h three times with shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
2. Dilute FITC-conjugated α-SMA antibody and purified rat anti-mouse CD31, which recognize smooth muscle actin on the artery to platelet endothelial cell adhesion molecule-1 on whole blood vessels, respectively, to 1:50 in staining buffer (see Recipe 5).
3. Immerse L1–L6 in 1.5 mL of the diluted antibody solution in a 5 mL polypropylene tube at room temperature for seven days with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
4. Wash L1–L6 with 40 mL of PBS in a 50 mL tube at room temperature for 30 min three times with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
5. For anti-CD31 staining:
  1. Further immerse L1–L6 in 1.5 mL of Alexa Fluor 555-conjugated goat anti-rat IgG diluted to 1:200 in staining buffer at room temperature for two days with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
  2. Wash L1–L6 with 40 mL of PBS in a 50 mL tube at room temperature for 30 min three times with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
6. Postfix L1–L6 with 10 mL of 1% PFA in PBS at room temperature for 5 h with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.
7. Wash L1–L6 with 40 mL of PBS at room temperature for 30 min three times with light shielding and shaking at 30 rpm in a seesaw motion on the TAITEC Wave-SI.

Refractive index adjustment
Living organisms are composed of molecules with different refractive indices, such as water, proteins, and lipids. In order to observe mammalian tissues in 3D by light sheet microscopy, it is important to suppress light scattering by making the refractive index uniform.
1. Immerse L1–L6 in 10 mL of 1/2 CUBIC-R [diluted CUBIC-R (see Recipe 6) to 1:1 in dH₂O] at room temperature for 5 h with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
2. Discard 1/2 CUBIC-R and immerse L1–L6 in 10 mL of CUBIC-R at room temperature overnight with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30.
3. Replace with fresh CUBIC-R and further immerse L1–L6 at room temperature for 4 h with light shielding and shaking at 100 rpm in an orbital motion on the TAITEC double shaker NR-30 (Figure 2).
  
  Figure 2. Cleared lumbar cords. Brightfield image of the lumbar cords before and after tissue clearing.

Microscopy and image analysis
1. Wash L1–L6 with 10 mL of observation oil (see Recipe 7).
2. Place L1–L6 into a glass container filled with observation oil.
3. Acquire 3D images using a light sheet microscope (UltraMicroscope II) as reported previously (Dodt et al., 2007; Susaki et al., 2014).
4. Collect all raw image data in lossless 16-bit TIFF format.
5. Visualize, capture, and analyze 3D-rendered images using Imaris software, version 9.0.0 (Figures 3 and 4, Videos 2 and 3).
  
  Figure 3. Detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of the artery with anti-SMA antibody. (A) aEAE mice with clinical score 2.5 and naïve mice were injected i.v. with Au₁₃ nanoclusters or solvent. Figures show cleared L3–L6 from each mouse stained with anti-SMA antibody, which recognizes smooth muscle actin on the artery. The signal of Au₁₃ nanoclusters was visualized as a punctate signal with a diameter of 50–200 nm in the dorsal blood vessels centered on L5 and in the parenchyma of the spinal cords in aEAE mice. Intravascular Au signals were also observed in aEAE and naïve mice injected with Au₁₃ nanoclusters. Scale bars, 1,500 μm. Magenta: Au₁₃ nanocluster; green: SMA (artery). (B) Magnified images of L5 from the aEAE and naïve mice in (A). Scale bars, 1,000 μm. Magenta: Au₁₃ nanocluster; green: SMA (artery).
  
  Figure 4. Detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of whole blood vessels with anti-CD31 antibody. aEAE mice with clinical score 3 were injected i.v. with Au₁₃ nanoclusters. Cleared L5 stained with anti-CD31 antibody, which recognizes platelet endothelial cell adhesion molecule-1 on whole blood vessels, is shown. Scale bars, 400 μm. Green: Au₁₃ nanocluster; red: CD31 (blood vessel).
  
  Video 2. The detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of the artery with anti-SMA antibody. A 3D movie of the lumbar spinal cords of an aEAE mouse with clinical score 2.5 after injecting i.v. with Au₁₃ nanoclusters. The cleared lumbar spinal cords were stained with anti-SMA antibody, which recognizes smooth muscle actin on the artery. Magenta: Au₁₃ nanocluster; green: SMA (artery).
  
  Video 3. The detection of microinflammation at L5 by Au₁₃ nanoclusters and staining of whole blood vessels with anti-CD31 antibody.A 3D movie of the lumbar spinal cords of an aEAE mouse with clinical score 3 after injecting i.v. with Au₁₃ nanoclusters. The cleared lumbar spinal cords were stained with anti-CD31 antibody, which recognizes platelet endothelial cell adhesion molecule-1 on whole blood vessels. Green: Au₁₃ nanocluster; red: CD31 (blood vessel).

Notes

Sodium azide–containing staining buffer is classified as a particularly hazardous substance due to high acute toxicity, especially to dermis.
CUBIC-R and other reagents are safe reagents, so there is no need to prepare them under a chemical fume hood. Further, they are highly stable and not difficult to prepare.
The injection of 200 μL of PTx or Au₁₃ nanoclusters i.v. should be performed by slowly pressing the insulin syringe and injecting the full volume of solution for approximately five seconds.
In our aEAE induction protocol, the disease usually develops with clinical scores of 2–3 on days 16–20 after the immunization. However, the timing of the disease onset and when the clinical score reaches 2–3 may depend on the manufacturing lot of the MOG peptides, CFA, and PTx as well as the mouse age and sex.
Mice with clinical score 3–4 were humanely euthanized according to ethical regulations.
When Au₁₃ nanoclusters were injected for 4 h, the Au signal was not detected. Therefore, we set the time to 2 h.
Mice were anesthetized with 4% isoflurane and PBS was then perfused while the mice were alive. Since the end of the transcardial perfusion uses 25 mL of PBS (more than 10 times the total blood volume), the mice were dead before the PFA perfusion.
In the fixation, washing, and refractive index adjustment steps, the entire tissue sample may be transferred from a decanter to a clean container and then transferred again into a tube filled with fresh buffer or solution using tweezers without a hook or spoon to minimize damage.
In step D2, L1–L6 should be excised from the spine carefully to minimize damage using micro-scissors and angled serrated tip forceps under the stereomicroscope with illuminator (Video 1).
The compounds used for delipidation/decolorization are surfactants and may affect antigenicity.
The lumbar spinal cords are regularly immersed in delipidation/decoloration buffer only once.
From step E3 onward, the samples should be protected from light.
For step E3, a 1.5 mL Eppendorf tube may be a bit small. For the spinal cord, a 2 mL tube is fine for antibody staining.
For light sheet microscopy, pictures are taken so that all tissues are included. It is recommended to acquire data in Z-stacks every 5 μm.
In our protocol, the spots were not counted. However, it would be possible to count visually or calculate the volume of each spot with a software such as Imaris.

Recipes

[Au₁₃(dppe)₅Cl₂]Cl₃ (Au₁₃)
Synthesize and isolate as crystalline solids according to the method of Shichibu and Konishi (2010).
1. Add 10 mL of ethanol containing NaBH₄ (66.2 mg, 1.75 mmol) to 240 mL of dichloromethane containing Au₂(dppe)Cl₂(302 mg, 0.35 mmol).
2. Stir the mixture at room temperature for 3 h.
3. Remove the solvent, suspend the residue in dichloromethane, and filter.
4. Evaporate the filtrate to dryness until forming a dark-brown powder (283.6 mg).
5. Dissolve the powder in ethanol (49 mL) and then treat with 1 mL aqueous hydrochloric acid (12 mmol).
6. Stir the mixture at room temperature for 24 h.
7. Evaporate the volatiles.
8. Wash the resulting residue with acetone.
9. Suspend the resulting residue in methanol and filter.
10. Evaporate the filtrate to dryness until forming [Au₁₃(dppe)₅CI₂]CI₃ (Au₁₃) as crystalline red solids (75.9 mg).
11. Weigh the crystalline solids of the Au₁₃ cluster (~3 mg).
12. Dissolve the crystalline solids in dimethyl sulfoxide (final concentration: 160 μM).
13. Store the nanoclusters at room temperature for up to one month with light shielding.

1 M HEPES buffer, pH 7.2 (1,000 mL)
1. Add 238.3 g of HEPES to 800 mL of dH₂O.
2. Adjust the buffer to pH 7.2 with 10 N NaOH.
3. Add dH₂O up to 1,000 mL (final concentration: 1 M).
4. To prepare 10 mM HEPES buffer, 1 M HEPES buffer is diluted 100-fold with dH₂O.
5. Store the buffer at room temperature for up to one month.

PBS (1,000 mL)
1. Add the following substances to 800 mL of dH₂O:
  8 g of NaCl (final concentration: 137 mM)
  0.2 g of KCl (final concentration: 2.68 mM)
  1.15 g of Na₂HPO₄ (final concentration: 8.1 mM)
  0.2 g of KH₂PO₄ (final concentration: 1.47 mM)
2. Add dH₂O up to 1,000 mL.
3. Store PBS at room temperature for up to one month.

Delipidation/decoloration buffer (500 g)
1. Add the following substances to 440 g of PBS:
  50 g of 1,2-Hexanediol [final concentration: 10% (w/w)]
  10 g of 1-Methylimidazole [final concentration: 2% (w/w)]
2. Store the buffer at room temperature for up to one month.

Staining buffer (100 mL)
1. Mix the following substances and add dH₂O up to 100 mL:
  5 mL of 10% Triton X-100 in dH₂O (final concentration: 0.5%)
  0.1 mL of 10% sodium azide in dH₂O (final concentration: 0.01%)
  5 mL of 20× PBS (final concentration: 1×)
  50 mL of 1% casein in PBS (final concentration: 0.5%; should be resolved to make 1% solution in PBS by warming at 60 °C)
2. Store the buffer at 4 °C for up to one month.

CUBIC-R (500 g)
1. Add the following substances to 125 mL dH₂O:
  225 g of 2,3-Dimethyl-1-phenyl-5-pyrazolone [final concentration: 45% (w/w)]
  150 g Nicotinamide [final concentration: 30% (w/w)]
2. Store CUBIC-R at room temperature for up to one month with light shielding.

Observation oil (100 g; refractive index = 1.52, 25 °C)
1. Add the following substances to a 100 mL tube and shake gently:
  60.2 g of silicone oil (HIVAC-F4) (Refractive index = 1.555, 25 °C)
  39.8 g of mineral oil (Refractive index = 1.467, 25 °C)
2. Measure the refractive index using a refractometer and adjust by adding either oil to 1.52.
3. Store the oil at room temperature for up to one month.

Acknowledgments

We appreciate the excellent technical assistance and secretarial assistance provided by Ms. C. Nakayama, Ms. S. Takano, Ms. S. Fukumoto, Ms. N. Yamamoto, Ms. S. Morita, and Ms. M. Ohsawa. We also thank Dr. Peter Karagiannis for proofreading the text. This work was supported by KAKENHI (MM, HR, SH, SIK), Japan Science and Technology Agency (JPMXS0120330644 (Q-Leap)), Japan Agency for Medical Research and Development (AMED) (MM; JP21zf0127004 and JP223fa627005), and the Takeda Science Foundation (MM, SH). This study was also supported partly by the Grant for Joint Research Program of the Institute for Genetic Medicine, Hokkaido University, by the Photo-Excitonix Project, Hokkaido University, and by the Promotion Project for Young Investigators at Hokkaido University (MM). This protocol is based on our previous experimental procedures for EAE induction (Arima et al., 2012; Ogura et al., 2008; Tanaka et al., 2017), Au₁₃ nanocluster preparation (Shichibu and Konishi, 2010), and CUBIC tissue-clearing protocol (Tainaka et al., 2014).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments described herein were approved by the Institutional Animal Care and Use Committees of the Institute for Genetic Medicine, Hokkaido University, and were performed according to institutional guidelines and regulations.

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