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

Published: April 30, 2024

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 Perform perfusion fixation following the steps below. 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 Tab…

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. <img alt="Figure 2" …

Divulgazioni

The authors have nothing to disclose.

Materials

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

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Citazione di questo articolo
A Whole-Mount Organ Staining Technique for Three-Dimensional Imaging of the Kidney. J. Vis. Exp. (Pending Publication), e22110, doi: (2024).

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