Whole Brain Clearing and Imaging Protocol using CUBIC and ZEISS Lightsheet Z.1

Whole Brain Clearing and Imaging Protocol using CUBIC and ZEISS Lightsheet Z.1

A. Tissue Clearing Using CUBIC

1. Fixation
   • Immerse the brain in 4% paraformaldehyde (PFA) in 1X PBS (pH 7.5).
   • Fix at 4 °C for 24 hours.
2. PBS Wash
   • Wash the brain in 1X PBS for 24 hours at 4 °C to remove excess fixative.
3. Delipidation with CUBIC-R1
   • Place the brain in CUBIC Reagent-1 (R1).
   • Incubate at 37 °C for 42 hours with gentle shaking, protected from light.
   • Note: Clearing speed varies with sample thickness and temperature.
4. Embedding in Low-Melting Agarose
   • Prepare 1% low-melting-point agarose in H₂O.
   • Cut off the tip of a 1 mL syringe.
   • Gently draw the brain into the syringe filled with molten agarose.
   • Allow the agarose to solidify at room temperature or 4 °C.
5. Refractive Index Matching in CUBIC-R2
   • Once solidified, submerge the agarose-embedded brain in CUBIC Reagent-2 (R2).
   • Store at 4 °C, protected from light, until imaging.

B. Lightsheet Imaging on ZEISS Lightsheet Z.1 Microscope

1. Sample Mounting
   • Insert the agarose-embedded brain (still in the cut syringe or removed and trimmed) into the ZEISS Z.1 sample holder.
   • Ensure the imaging chamber is filled with CUBIC-R2 (RI ≈ 1.49), which is compatible with the Z.1's detection and illumination optics.
2. System Setup (ZEN Black or ZEN Lightsheet Software)
   • Detection Objective: 5×.
   • Laser lines: Choose based on fluorophores (e.g., 488 nm for GFP, 561 nm for tdTomato, 640 nm for far-red dyes).
   • Light sheet thickness: Optimize to balance resolution and speed (e.g., 4–8 µm).
   • Z-step size: Typically 2–5 µm depending on optical sectioning needs.
   • Exposure time: 50–150 ms per plane (optimize based on signal intensity).
3. Imaging Options
   • Use multiview imaging (e.g., 0° and 180°) if sample opacity remains or for higher-resolution reconstruction.
   • Use automated z-stack acquisition to capture the entire volume.