Cryoprotection

1. Remove PBS-T from organoids and discard. Add 5 mL 30% sucrose solution per organoid.
2. Allow samples to equilibrate in 30% sucrose solution overnight at 2 – 8°C.
   Time to equilibrate can vary due to organoid size and density. Once organoids no longer float in the 30% sucrose solution, it is appropriate to move to the next step.

Embedding

Gelatin is preferable for neural organoids over other embedding reagents such as optimal cutting temperature compound (OCT), as it provides better rigidity and supports the generation of smooth, clean sections. Gelatin will begin to polymerize and harden at room temperature, so it is necessary to quickly transfer organoids to the embedding mold.

A thin layer of gelatin embedding solution may first be added to the mold and allowed to solidify prior to adding the organoid. This will allow the organoid to be positioned more easily toward the center of the block. Once the organoid has been placed in the mold and the embedding solution has been topped up, use a pipette tip to reposition the organoid if needed.

1. Warm gelatin solution to 37°C in a water bath.
2. Pipette sucrose solution out of conical tube and discard. Add enough gelatin solution to completely cover organoids.
   Multiple organoids may be embedded in a single block if desired. On the other hand, older or larger organoids may have necrotic centers, which means they can shear more easily during sectioning. These samples can benefit from being embedded in larger gelatin blocks to provide more external support.
3. Incubate at 37°C for 1 hour. This allows the gelatin to penetrate and encapsulate the organoid.
   For older or larger organoids, additional incubation time with liquid gelatin at 37°C before embedding can also help by allowing the gelatin to better penetrate the tissue and provide support.
4. Remove organoids from conical tube and transfer to embedding mold.

Snap Freezing

Rapid freezing helps to prevent the formation of ice crystals in the organoids and to maintain the native cellular architecture of the sample.

1. Prepare a dry ice/ethanol slurry by adding dry ice to 100% ethanol.
2. Once the mixture stops boiling, add the embedded sample by holding the embedding mold with forceps and submerging it into the slurry.
3. Keep the sample in the cold slurry until completely frozen, then transfer to a -80°C freezer for long-term storage.
   If freezing artifacts are still present in samples, snap freezing can also be done via immersion into an isopentane bath chilled with liquid nitrogen. Alternatively, samples may be placed directly into liquid nitrogen in an appropriate vessel such as a dewar flask.

Cryosectioning

1. Remove blocks from the -80°C freezer and allow them to warm to sectioning temperature in the cryostat for 30 minutes.
   • Optimal sectioning temperature for gelatin-embedded organoids is -26 to -30°C.
   • Typical sectioning thickness is 10 – 20 μm, and can be adjusted based on the imaging to be performed.
   • Collection of multiple serial sections can allow exploration of different markers in different sections.
   • If sections are curling, use a thin brush that has been cooled and kept in the cryostat chamber to gently flatten the section before mounting it on the slide.
   • Always use a new, sharp blade for sectioning.