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).