Whole-Kidney Three-Dimensional Staining with CUBIC

Summary

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.

Abstract

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

Introduction

The kidney is composed of various cell populations. Although conventional pathology gives us much information about the kidney microenvironment, three-dimensional (3D) imaging is needed to precisely understand the intercellular crosstalk during kidney disease progression. In the past, a huge number of serial sectioning and image reconstruction needed to be performed for the whole-organ 3D imaging¹. However, this method required too much effort and had problems in terms of reproducibility.

Optical clearing is a good strategy to overcome this hurdle^2,3. Tissue opacity is mainly due to light scattering and absorption because each organ consists of various substances, including water, protein, and lipids. Thus, the basic strategy of tissue clearing is replacing water and lipids in tissues with refractive index (RI) matching reagents that have the same optical properties as proteins⁴. In order to observe a transparent specimen, a light sheet fluorescent microscopy is useful⁵. Light sheets illuminate the transparent specimen from the side, and excitation signals are acquired through the objective lens located in a vertical position⁶. This microscopy obtains cross-section information in a single sweep, which is different from the confocal or multiphoton fluorescent microscopy. Thus, it can quickly acquire z-stack images with a low level of photobleaching.

Clear, Unobstructed Brain/Body Imaging Cocktails and Computational Analysis (CUBIC) is one of the tissue clearing methods which enables whole-organ imaging by light sheet fluorescent microscopy^2,7,8. CUBIC and whole-mount immunofluorescent staining are optimized in the present study to visualize mouse kidney 3D structures^9,10,11. Using this whole-mount staining method, the alteration in renal sympathetic nerves is visualized after ischemia-reperfusion injury^9,10 and glomerulomegaly in the early stage of diabetic kidney disease¹¹, as well as blood vessels, proximal tubules, and collecting ducts in a whole kidney⁹.

Protocol

All experiments were approved by the University of Tokyo Institutional Review Board. All animal procedures were performed according to the National Institutes of Health guidelines. Male C57BL/6NJcl mice, 8 weeks old, were used for the present study. The mice were obtained from commercial sources (see Table of Materials).

1. Animal preparation and kidney fixation

1. Perform perfusion fixation following the steps below.
   1. 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).
   2. Perfuse the animal with 20 mL of PBS (pH 7.4) and 30 mL of 4% PFA in phosphate buffer (PB) through the left ventricle of the heart¹².
2. Perform the immersion fixation just after the perfusion fixation and kidney sampling⁹.
   1. Immerse the kidney in 4% PFA at 4 °C for an additional 16 h, then wash it with PBS for 2 h (three times)⁹ (Figure 1).
      ​CAUTION: Formaldehyde and paraformaldehyde are toxic irritants. Handle reagents in a fume hood with appropriate personal protective equipment.

2. Decolorization and delipidation

1. Prepare CUBIC-L 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^4,8,9.
2. Perform decolorization and delipidation by CUBIC-L following the steps below.
   1. 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 °C for 5 days.
   2. 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)⁹ (Figure 1). Use a dispensing spoon for sample handling (Figure 2B).

3. Whole-mount immunofluorescent staining

1. Prepare the staining buffers.
   1. 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 azide⁹.
   2. 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 azide⁹.
2. Perform staining with primary antibodies.
   1. Immunostain the delipidated kidney with primary antibodies (1:100 or 1:200, see Table of Materials) in the staining buffer at 37 °C with gentle shaking for 7 days.
      NOTE: The amount of the staining buffer needed for one kidney is 500-600 µL (Figure 2C).
   2. Wash the kidney with 0.5% (v/v) Triton X-100 in PBS (PBST) at room temperature for 1 day⁹ (Figure 1).
3. Perform staining with secondary antibodies.
   1. Immunostain the kidney with secondary antibodies (1:100 or 1:200, see Table of Materials) in the staining buffer at 37 °C with gentle shaking for 7 days. Wash the kidney with PBST at room temperature for 1 day⁹ (Figure 1).
   2. Post fixation⁹, 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).

4. Refractive index (RI) matching

1. Prepare CUBIC-R+.
   1. 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).
   2. Prepare CUBIC-R+ for refractive index (RI) matching by adding 0.5 v% of N-buthyldiethanolamine to CUBIC-R following the previously published reports^4,8,9.
2. Perform the RI matching.
   1. 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 days⁹ (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).

5. Image acquisition and reconstruction

1. 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)⁵ (Figure 3).
2. Acquire 3D images of the whole kidney with a custom-built⁹ 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).

Results

Using this staining method, sympathetic nerves [anti-tyrosine hydroxylase (TH) antibody] and arteries [anti-α-smooth muscle actin (αSMA) antibody] in a whole kidney (Figure 4A,B and Video 1) were visualized⁹. Abnormal renal sympathetic nerves were also visualized after ischemia/reperfusion injury (IRI)^9,10 (Figure 4C). Moreover, visualizing sympat...

Discussion

The present protocol allowed whole-kidney 3D imaging of various structures such as sympathetic nerves, collecting ducts, arteries, proximal tubules, and glomeruli^9,10,11. This staining method offered macroscopic observation and led to new biological discoveries, by visualizing the alteration in renal sympathetic nerves after ischemia-reperfusion injury^9,10 and glomerulom...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
14 mL Round Bottom High Clarity PP Test Tube	Falcon	352059	Tissue clearing, staining, wash
2,3-dimethyl-1-phenyl-5-pyrazolone/antipyrine	Tokyo Chemical Industry	D1876	CUBIC-R+
37%-Formaldehyde Solution	Nacalai Tesque	16223-55	Post fixation
4%-Paraformaldehyde Phosphate Buffer Solution	Nacalai Tesque	09154-85	Kidney fixation
Alexa Flour 555-conjugated donkey anti-sheep IgG antibody	Invitrogen	A-21436	Secondary antibody (1:100)
Alexa Flour 647-conjugated donkey anti-rabbit IgG antibody	Invitrogen	A-31573	Secondary antibody (1:200)
Anti-aquaporin 2 (AQP2) antibody	Abcam	ab199975	Primary antibody (1:100)
Anti-podocin antibody	Sigma-Aldrich	P0372	Primary antibody (1:100)
Anti-sodium glucose cotransporter 2 (SGLT2) antibody	Abcam	ab85626	Primary antibody (1:100)
Anti-tyrosine hydroxylase (TH) antibody	Abcam	ab113	Primary antibody (1:100)
Anti-α-smooth muscle actin (α-SMA) antibody	Abcam	ab5694	Primary antibody (1:200)
Blocker Casein in PBS	Thermo Fisher Scientific	37528	Staining buffer
Butorphanol Tartrate	Meiji	005526	Anesthetic
C57BL/6NJcl	Nippon Bio-Supp.Center	N/A	Mouse strain
Imaris	Bitplane	N/A	Imaging analysis software
Macro-zoom microscope	OLYMPUS	MVX10	The observation unit of the custom-built microscope
Medetomidine Hydrochloride	Kyoritsu-Seiyaku	008656	Anesthetic
Midazolam	SANDOZ	27803229	Anesthetic
Mineral oil	Sigma-Aldrich	M8410	Immersion oil
N-buthyldiethanolamine	Tokyo Chemical Industry	B0725	CUBIC-L, CUBIC-R+
Nicotinamide	Tokyo Chemical Industry	N0078	CUBIC-R+
Polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100	Nacalai Tesque	12967-45	CUBIC-L, PBST
Silicon oil HIVAC-F4	Shin-Etsu Chemical	50449832	Immersion oil
Sodium azide	Wako	195-11092	Staining buffer