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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 Table of Materials).
- 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.
- Perform the immersion fixation just after the perfusion fixation and kidney sampling.
- 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
- 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.
- Perform decolorization and delipidation by CUBIC-L following the steps below.
- 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.
- 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
- Prepare the staining buffers.
- 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.
- 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.
- Perform staining with primary antibodies.
- 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).
- Wash the kidney with 0.5% (v/v) Triton X-100 in PBS (PBST) at room temperature for 1 day (Figure 1).
- Perform staining with secondary antibodies.
- 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).
- 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
- Prepare CUBIC-R+.
- 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).
- Prepare CUBIC-R+ for refractive index (RI) matching by adding 0.5 v% of N-buthyldiethanolamine to CUBIC-R following the previously published reports.
- Perform the RI matching.
- 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
- 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).
- 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).
