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.
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.
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 hurdle2,3. 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 microscopy2,7,8. CUBIC and whole-mount immunofluorescent staining are optimized in the present study to visualize mouse kidney 3D structures9,10,11. Using this whole-mount staining method, the alteration in renal sympathetic nerves is visualized after ischemia-reperfusion injury9,10 and glomerulomegaly in the early stage of diabetic kidney disease11, as well as blood vessels, proximal tubules, and collecting ducts in a whole kidney9.
The present protocol allowed whole-kidney 3D imaging of various structures such as sympathetic nerves, collecting ducts, arteries, proximal tubules, and glomeruli9,10,11. This staining method offered macroscopic observation and led to new biological discoveries, by visualizing the alteration in renal sympathetic nerves after ischemia-reperfusion injury9,10 and glomerulom…
The authors have nothing to disclose.
Part of this work was conducted through collaboration with Prof. Hiroki R. Ueda (University of Tokyo), Prof. Etsuo A. Susaki (Juntendo University), Prof. Tetsuhiro Tanaka (Tohoku University), Prof. Masafumi Fukagawa, Dr. Takehiko Wada, and Dr. Hirotaka Komaba (Tokai University).
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 |