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

Abstract

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 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) ....

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 .......

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	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