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

Overview

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.

Protocol

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

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 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.
2. Perform the immersion fixation just after the perfusion fixation and kidney sampling.
   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 (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.
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) (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 azide.
   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 azide.
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 day (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 day (Figure 1).
   2. 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).

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.
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 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).

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) (Figure 3).
2. 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).

Representative Results

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.

Figure 2: Images of the handling process. (A) The sample is immersed in 7 mL of 50% or 100% CUBIC-L in a 14 mL round bottom tube. The tube is kept horizontal with gentle shaking during the delipidation process. (B) A dispensing spoon is used for sample handling. (C) The amount of the staining buffer needed for one kidney is 500-600 µL. The tube is kept vertical during the staining process. (D) Although the sample 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 3: Light sheet fluorescent microscopy for whole-kidney 3D imaging. Light sheet fluorescent microscopy (LSFM) enables three-dimensional (3D) imaging of transparent samples. This image is reproduced from Hasegawa et al.9. Immersion oil is used during data acquisition. Light sheets from both sides illuminate the sample. The excitation signals are acquired through the objective lens located in a vertical position. The stage moves in the axial direction, and z-stack images are obtained. Scale bar = 10 mm.

Materials

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	5526	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	8656	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