Materials and reagents

Biological materials

Murky tissues previously cleared by iDISCO and possibly other hydrophobic clearing methods

Reagents

Methanol (Sigma-Aldrich, catalog number: 179337)
Dichloromethane (Sigma-Aldrich, catalog number: 270997)
Dibenzyl ether (Sigma-Aldrich, catalog number: 33630)

Solutions

Dichloromethane and methanol mix (see Recipes)

Recipes

Dichloromethane and methanol mix (per sample)
Reagent Final concentration (vol%) Volume
Dichloromethane 66% 3.3 mL
Methanol 34% 1.7 mL
Total n/a 5.0 mL
The mix should be made freshly and kept at room temperature before use.

Laboratory supplies

Organ holding forceps (e.g., Fine Science Tools, catalog number: 11031-15)
Screw-cap glass vials (e.g., Millipore Sigma, catalog number: 27151)
Nitrile gloves (any brand)
Kimwipes and paper towels (any brand)

Equipment

Fume hood (e.g., Hamilton Lab, model: SafeAire II)
Orbital shaker (e.g., Stovall Life Science, Belly Dancer, model: BDRAA115S)
Light-sheet microscope (e.g., Miltenyi Biotec, model: UltraMicroscope II Blaze)
Computer workstation equipped with, at least, Intel Xeon CPU, 128 G RAM, GPU with 16 GB memory, 2 TB storage, and USB3.0 port.

Software and datasets

Cloud-based lab management software (e.g., Labguru.com) is recommended to track and record the rescue procedure
Microscope system control and image acquisition software (e.g., ImSpector 7.5.3, free)
Image processing software (e.g., ImageJ1.53, free)
Atlas-matching and immunofluorescence quantification software (e.g., NeuroInfo, licensed by MBF Bioscience)

Procedure

Dehydration
Note: Please see note 2 and 3 about waste disposal and light avoidance.
1. Transfer the murky tissue sample (Figure 1) to a 5 mL screw-cap glass vial filled with 100% methanol and seal tightly.
  
  Figure 1. Two previously cleared but murky brain samples over the course of rescue. Brain hemisphere (IDT3WT) and whole brain (S3) previously cleared by iDISCO but that turned murky during months of storage. Samples in A) and B) become totally opaque after dehydration; in C) they remain totally opaque after delipidation, with transparency restored with a light-yellow tinge, D) after refractive index (RI) matching.
2. Place the vial on an orbital shaker (e.g., Belly Dancer) and shake at 70 rpm at room temperature for 1 h.
3. Discard methanol and repeat steps A1 and A2 three more times.
Delipidation
1. Transfer tissue sample (Figure 1B) to a 5 mL screw-cap glass vial filled with 66% dichloromethane + 34% methanol and seal tightly.
  CAUTION: Wear double nitrile gloves and work in a ventilation hood when handling dichloromethane. Wipe the exterior of the vials with paper towels.
2. Place the vial on an orbital shaker and shake at 70 rpm at room temperature for 48 h.
3. Transfer tissue sample to a 5 mL screw-cap glass vial filled with 100% dichloromethane and seal tightly.
  CAUTION: One can always keep the sample in the same vial and change the solution instead (see note 4).
4. Place the vial on an orbital shaker and shake at 70 rpm at room temperature for 1 h.
RI rematching
1. Transfer tissue sample (Figure 1C) to a 5 mL screw-cap glass vial filled with 100% dibenzyl ether and seal tightly.
  CAUTION: Wear double nitrile gloves and work in a ventilation hood when handling dibenzyl ether. Wipe the exterior of the vials with paper towels.
2. Place the vial on an orbital shaker and shake at 70 rpm at room temperature for 48 h.
Storage
1. Transfer tissue sample (Figure 1D) to a 5 mL screw-cap glass vial filled with fresh 100% dibenzyl ether and seal tightly.
  CAUTION: Ensure no air bubbles are visible in the storage vials, which can be done by slight overfill of the glass vial and closing the cap very slowly.
2. Store the sample at room temperature and refresh dibenzyl ether once a month.

Data analysis

Tissue samples are imaged by light-sheet microscopy (e.g., Blaze) to localize immunofluorescence. Compiling of Z-stack and stitching of tiles is carried out using image processing software (e.g., Imaris). Atlas-matching and immunofluorescence measuring are achieved by image analysis programs (e.g., NeuroInfo). Figure 2A shows fluorescence light-sheet images of mouse brain before and after rescue. Figure 2B summarizes the fluorescence changes in different brain regions before and after rescue.

Figure 2. Rescued mouse brain. A. Sample images before and after the rescue. Overlay images are the composite of CF568, 640, and 770 signals. CF568 images show an apparent haze in midbrain and thalamus (before), which was cleared after rescue. Scale bar, 2 mm. B. Average fluorescence for all three immunofluorescent labels (arbitrary unit, a.u.; calculated from 15–30 randomly selected regions of interest within those brain regions). The increases are likely due to significantly lowered background (i.e., much less diffraction) after the rescue.

Validation of protocol

We have applied this rescue protocol to three different batches of murky brain tissues previously cleared by iDISCO and two more batches of murky brain tissues similarly cleared by a different researcher from another lab. In total, we tested this protocol using eight murky brain samples and successfully re-cleared all of them while preserving immunofluorescent signal.

General notes and troubleshooting

Unless specified, all steps are carried out at room temperature.
Waste disposal. As with initial iDISCO clearing, users should follow institutional requirements for waste disposal as established by local Environmental Health and Safety officers. All reagents used in this protocol should be disposed of in appropriate liquid chemical waste containers.
Light avoidance. If the tissue samples to be rescued have immunofluorescence labels, caution is needed to reduce loss of fluorescence. Options include always using aluminum foil to cover the glass vials containing tissue samples and operating under reduced light.
Solution change. Although the protocol above describes solution change as involving a transfer of samples to new vials containing the needed solutions, one can also discard the solution with retention of the tissue in the original vials, followed by adding new solutions to the original vial. It is important that containers (i.e., glass vials) holding samples are completely filled with solutions.
This rescue protocol is tested for the rescue of iDISCO-cleared samples. In rescued mouse brains, we used the following primary antibodies: one is the combination of guinea pig anti-GFAP antibody, chicken anti-Tuj-1 antibody, and rabbit anti-SREBP2 antibody; the other is rabbit anti-cFos alone. Accordingly, the secondary antibodies are highly cross-absorbed CF770-conjugated goat anti-guinea-pig IgG, CF640-conjugated goat anti-chicken IgG, and CF-568-conjugated goat anti-rabbit IgG (all from Biotium).
The timeline of this protocol is adjustable depending on the size of the samples.
Like iDISCO, the survival of different epitopes as well as corresponding antibodies during the rescue needs to be evaluated on a case-by-case basis. Since the rescue procedure and solutions used are largely the same to those of iDISCO, it is likely that antibodies suitable for iDISCO should be compatible with this rescue procedure.
Because this protocol is based on the hydrophobic iDISCO method, it may also work for samples prepared by other hydrophobic clearing methods but not for samples cleared by hydrophilic methods.

Acknowledgments

H.M., J.M., and Q.Z. were supported by NIH award GM147912, a Florida Department of Health award 21A04, and a pilot award from the Stiles-Nicholson Brain Institute. P.G.-K. and R.B. were supported by NIH Award MH094527.

Competing interests

The authors declare no competing interests.

Ethical considerations

Animal use and related procedures were approved by the Florida Atlantic University Institutional Animal Care and Use Committee (IACUC Protocol #A21-06). Mice were housed in pairs. A total of eight male and female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) between the age of 6 and 30 months were used for collecting brain tissues for this study.

References

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