Hi,

Yes, several groups have successfully performed whole mouse brain clearing for light sheet 3D microscopy, but it is true that the process can be challenging, especially when trying to maintain endogenous fluorescence while achieving deep antibody penetration. Based on published protocols and practical experience, here are some recommendations:

1. Clearing Protocol:

• iDISCO+ is widely used for antibody labeling in cleared mouse brains. The “+” version includes modifications to reduce background autofluorescence and improve penetration.
• uDISCO is another method that preserves endogenous fluorescence better than iDISCO and reduces tissue shrinkage.
• CUBIC and SHIELD are also effective for preserving endogenous fluorescent proteins while allowing immunolabeling, though antibody penetration may still require long incubation times.

Key tips to improve results:

• Autofluorescence reduction: Pre-treat the brain with 0.1–0.5% hydrogen peroxide in methanol overnight to reduce autofluorescence.
• Antibody penetration: Use prolonged incubation times (several days to a week) at 37°C with gentle shaking; consider adding 0.3–0.5% Triton X-100 and 5–10% DMSO to enhance tissue permeability.
• Blocking and washing: Ensure thorough blocking with serum and detergents, and perform extended washes to reduce background signal.
• Registration & quantification: For registration, software like ClearMap, Imaris, Arivis Vision4D, or elastix/Fiji plugins can help align cleared brains to atlases (e.g., Allen Brain Atlas). For quantification, volumetric cell counting with Imaris or ClearMap is commonly used.

Microscopy:

• Most groups use light sheet fluorescence microscopy (LSFM), such as LaVision UltraMicroscope II or Zeiss Lightsheet Z.1, for whole-brain imaging. These systems allow imaging of large volumes at high resolution.
• Optimize excitation and emission settings to reduce bleed-through and autofluorescence; for example, longer wavelength dyes often give cleaner signals.

Maintaining endogenous fluorescence:

• Avoid excessive methanol dehydration or long exposure to harsh solvents; protocols like uDISCO or SHIELD are better for preserving fluorescent proteins.
• Some studies combine endogenous GFP signal with antibody staining using mild clearing methods and post-staining refractive index matching.

References/Protocols:

• Renier, N., et al., Cell 2014; 159:896–910 (iDISCO).
• Susaki, E.A., et al., Cell 2014; 157:726–739 (CUBIC).
• Pan, C., et al., Nat. Protoc. 2016; 11:222–238 (uDISCO).
• Yang, B., et al., Nat. Neurosci. 2014; 17:507–513 (ClearMap for quantification).

Overall, successful whole-brain imaging requires patience, careful optimization of antibody penetration, and appropriate clearing method based on whether you prioritize antibody labeling or endogenous fluorescence.

Zein Al-Abideen Douba thank you for your answer

Can you kindly share recent scientific publications using this technology - meaning that include quatified resuls achieved by light sheet 3d microscopy in a whole mouse brain.

IT WILL BE HIGHLY APPRECIATED

Adva Beck Certainly—here are several very recent scientific studies (from 2024–2025) that not only apply tissue clearing and light-sheet 3D microscopy to entire mouse brains, but also provide quantitative results based on imaging data: