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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal preparation and kidney fixation
<ol>
	<li>Perform perfusion fixation following the steps below.
	<ol>
		<li>Anesthetize the mouse by inhalation of isoflurane (3%, 2.0 L/min) and intraperitoneal administration of medetomidine hydrochloride (0.3 mg/kg), butorphanol tartrate (5 mg/kg), and midazolam (4 mg/kg) (see Table of Materials).</li>
		<li>Perfuse the animal with 20 mL of phosphate-buffered saline (PBS, pH 7.4) and 30 mL of 4% paraformaldehyde (PFA) in phosphate buffer (PB) through the left ventricle of the heart.</li>
	</ol>
	</li>
	<li>Perform the immersion fixation just after the perfusion fixation and kidney sampling.
	<ol>
		<li>Immerse the kidney in 4% PFA at 4 &#176;C for an additional 16 h, then wash it with PBS for 2 h (three times)9 (Figure 1). 
		&#8203;CAUTION: Formaldehyde and paraformaldehyde are toxic irritants. Handle reagents in a fume hood with appropriate personal protective equipment.</li>
	</ol>
	</li>
</ol>
2. Decolorization and delipidation
<ol>
	<li>Prepare CUBIC-L (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) for decolorization and delipidation consisting of 10 wt% of Triton X-100 and 10 wt% of N-buthyldiethanolamine (see Table of Materials), following previously published reports.</li>
	<li>Perform decolorization and delipidation by CUBIC-L following the steps below.
	<ol>
		<li>Immerse the fixed kidney in 7 mL of 50% (v/v) CUBIC-L (1:1 mixture of water and CUBIC-L) in a 14 mL round bottom tube (see Table of Materials) with gentle shaking at room temperature for 6 h (Figure 2A). Then, immerse it in 7 mL of CUBIC-L in a 14 mL round bottom tube with gentle shaking at 37 &#176;C for 5 days.</li>
		<li>Refresh CUBIC-L every day during this process. After the decolorization and delipidation process, wash the kidney with PBS at room temperature for 2 h (three times) (Figure 1). Use a dispensing spoon for sample handling (Figure 2B).</li>
	</ol>
	</li>
</ol>
3. Whole-mount immunofluorescent staining
<ol>
	<li>Prepare the staining buffers.
	<ol>
		<li>Prepare the staining buffer for primary antibodies by mixing 0.5% (v/v) Triton X-100, 0.5% casein in PBS, and 0.05% sodium azide.</li>
		<li>Prepare the staining buffer for secondary antibodies by mixing 0.5% (v/v) Triton X-100, 0.1% casein in PBS, and 0.05% sodium azide.</li>
	</ol>
	</li>
	<li>Perform staining with primary antibodies.
	<ol>
		<li>Immunostain the delipidated kidney with primary antibodies (1:100 or 1:200, see Table of Materials) in the staining buffer at 37 &#176;C with gentle shaking for 7 days. 
		NOTE: The amount of the staining buffer needed for one kidney is 500-600 &#181;L (Figure 2C).</li>
		<li>Wash the kidney with 0.5% (v/v) Triton X-100 in PBS (PBST) at room temperature for 1 day (Figure 1).</li>
	</ol>
	</li>
	<li>Perform staining with secondary antibodies.
	<ol>
		<li>Immunostain the kidney with secondary antibodies (1:100 or 1:200, see Table of Materials) in the staining buffer at 37 &#176;C with gentle shaking for 7 days. Wash the kidney with PBST at room temperature for 1 day (Figure 1).</li>
		<li>Post fixation, immerse the kidney in 1% formaldehyde in PB (1:36 mixture of 37% formaldehyde and PB) for 3 h, and wash it with PBS at room temperature for 6 h (Figure 1).</li>
	</ol>
	</li>
</ol>
4. Refractive index (RI) matching
<ol>
	<li>Prepare CUBIC-R+.
	<ol>
		<li>Prepare CUBIC-R by mixing 45 wt% of 2,3-dimethyl-1-phenyl-5-pyrazolone/antipyrine and 30 wt% of nicotinamide (see Table of Materials).</li>
		<li>Prepare CUBIC-R+ for refractive index (RI) matching by adding 0.5 v% of N-buthyldiethanolamine to CUBIC-R following the previously published reports.</li>
	</ol>
	</li>
	<li>Perform the RI matching.
	<ol>
		<li>Immerse the kidney in 7 mL of 50% (v/v) CUBIC-R+ (1:1 mixture of water and CUBIC-R+) in a 14 mL round bottom tube with gentle shaking at room temperature for 1 day. Then, immerse it in 7 mL of CUBIC-R+ in a 14 mL round bottom tube with gentle shaking at room temperature for 2 days (Figure 1). 
		NOTE: Although the kidney floats in 50% CUBIC-R+ at the beginning of the RI matching, it sinks and becomes transparent in CUBIC-R+ at the end of the process (Figure 2D).</li>
	</ol>
	</li>
</ol>
5. Image acquisition and reconstruction
<ol>
	<li>Immerse the RI-matched kidney in a mixture of silicon oil (RI = 1.555) and mineral oil (RI = 1.467) (55:45) during image acquisition (RI = 1.51) (Figure 3).</li>
	<li>Acquire 3D images of the whole kidney with a custom-built light-sheet fluorescent microscope (see Table of Materials). Collect all raw image data in a 16-bit TIFF format. Visualize and capture the 3D-rendered images with the imaging analysis software (Imaris, see Table of Materials).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>14 mL Round Bottom High Clarity PP Test Tube</td>
			<td>Falcon</td>
			<td>352059</td>
			<td>Tissue clearing, staining, wash</td>
		</tr>
		<tr>
			<td>2,3-dimethyl-1-phenyl-5-pyrazolone/antipyrine</td>
			<td>Tokyo Chemical Industry</td>
			<td>D1876</td>
			<td>CUBIC-R+</td>
		</tr>
		<tr>
			<td>37%-Formaldehyde Solution</td>
			<td>Nacalai Tesque</td>
			<td>16223-55</td>
			<td>Post fixation</td>
		</tr>
		<tr>
			<td>4%-Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Nacalai Tesque</td>
			<td>09154-85</td>
			<td>Kidney fixation</td>
		</tr>
		<tr>
			<td>Alexa Flour 555-conjugated donkey anti-sheep IgG antibody</td>
			<td>Invitrogen</td>
			<td>A-21436</td>
			<td>Secondary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Alexa Flour 647-conjugated donkey anti-rabbit IgG antibody</td>
			<td>Invitrogen</td>
			<td>A-31573</td>
			<td>Secondary antibody (1:200)</td>
		</tr>
		<tr>
			<td>Anti-aquaporin 2 (AQP2) antibody</td>
			<td>Abcam</td>
			<td>ab199975</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-podocin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>P0372</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-sodium glucose cotransporter 2 (SGLT2) antibody</td>
			<td>Abcam</td>
			<td>ab85626</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-tyrosine hydroxylase (TH) antibody</td>
			<td>Abcam</td>
			<td>ab113</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-&#945;-smooth muscle actin (&#945;-SMA) antibody</td>
			<td>Abcam</td>
			<td>ab5694</td>
			<td>Primary antibody (1:200)</td>
		</tr>
		<tr>
			<td>Blocker Casein in PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>37528</td>
			<td>Staining buffer</td>
		</tr>
		<tr>
			<td>Butorphanol Tartrate</td>
			<td>Meiji</td>
			<td>5526</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>C57BL/6NJcl</td>
			<td>Nippon Bio-Supp.Center</td>
			<td>N/A</td>
			<td>Mouse strain</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td>N/A</td>
			<td>Imaging analysis software</td>
		</tr>
		<tr>
			<td>Macro-zoom microscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td>The observation unit of the custom-built microscope</td>
		</tr>
		<tr>
			<td>Medetomidine Hydrochloride</td>
			<td>Kyoritsu-Seiyaku</td>
			<td>8656</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>SANDOZ</td>
			<td>27803229</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>Immersion oil</td>
		</tr>
		<tr>
			<td>N-buthyldiethanolamine</td>
			<td>Tokyo Chemical Industry</td>
			<td>B0725</td>
			<td>CUBIC-L, CUBIC-R+</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Tokyo Chemical Industry</td>
			<td>N0078</td>
			<td>CUBIC-R+</td>
		</tr>
		<tr>
			<td>Polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100</td>
			<td>Nacalai Tesque</td>
			<td>12967-45</td>
			<td>CUBIC-L, PBST</td>
		</tr>
		<tr>
			<td>Silicon oil HIVAC-F4</td>
			<td>Shin-Etsu Chemical</td>
			<td>50449832</td>
			<td>Immersion oil</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Wako</td>
			<td>195-11092</td>
			<td>Staining buffer</td>
		</tr>
	</tbody>
</table>

a whole-mount organ staining technique for three-dimensional imaging of the kidney

Source: Hasegawa, S., et al. <a href="https://app.jove.com/v/63986">Whole-Kidney Three-Dimensional Staining with CUBIC.</a> J. Vis. Exp. (2022).
This video demonstrates an assay for the whole-mount staining of murine kidneys using a tissue-clearing method called CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) and immunofluorescent staining. The kidney is subjected to tissue clearing, wherein the kidney is delipidated, decolorized, and treated with a refractive index matching solution to make the tissue transparent for optimum three-dimensional imaging. The tissue is stained with fluorescently labeled antibodies to reveal sympathetic nerves and arteries in the tissue under a light-sheet microscope.

<img alt="Figure 1" src="/files/ftp_upload/22110/22110_Figure1.jpg" /> 
Figure 1: Tissue clearing and whole-mount immunofluorescent staining. Tissue clearing by CUBIC is a two-step process: delipidation (CUBIC-L) and refractive index (RI) matching (CUBIC-R+). Whole-mount staining is performed after the delipidation process. Scale bar = 10 mm. The image is reproduced from Hasegawa et al.
<img alt="Figure 2" ...

A Whole-Mount Organ Staining Technique for Three-Dimensional Imaging of the Kidney

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

Take a fixed mouse kidney. Lipids in the tissue cause light scattering, and pigments cause light absorption, making the cells opaque.
Treat the tissue with CUBIC-L for delipidation and decolorization. Amino alcohol in CUBIC-L removes lipids and pigments, minimizing light scattering and increasing transparency.
Detergent molecules in CUBIC-L also permeabilize the cells.
Introduce a primary antibody cocktail that binds to cytoplasmic tyrosine hydroxylase in neurons of the sympathetic nerves and alpha-smooth muscle actin in vessel smooth muscle cells.
Add fluorophore-labeled secondary antibodies that bind to the primary antibodies.
Perform tissue postfixation to preserve the antibody treatment.
Immerse the tissue in CUBIC-R+ that replaces water and matches the refractive index of proteins, further minimizing light scattering and increasing transparency.
Under a light-sheet fluorescence microscope, light sheets illuminate the tissue, exciting the fluorophores.
Capture the fluorophore-emitted light and reconstruct a three-dimensional image, displaying the sympathetic nerve distribution around the arteries in the kidney.

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115 Views. Source: Hasegawa, S., et al. Whole-Kidney Three-Dimensional Staining with CUBIC. J. Vis. Exp. (2022). This video demonstrates an assay for the whole-mount staining of murine kidneys using a tissue-clearing method called CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) and immunofluorescent staining. The kidney is subjected to tissue clearing, wherein the kidney is delipidated, decolorized, and treated with a refractive index matching solution to make the tissu...

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A Whole-Mount Organ Staining Technique for Three-Dimensional Imaging of the Kidney

Transcript

A Whole-Mount Organ Staining Technique for Three-Dimensional Imaging of the Kidney

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids