Collecting larger-sized organoids: When grown in a 96-well plate with high density, use a pipette to remove more than half of the well, wash 1 to 3 times to remove most of the basement membrane matrix, centrifuge to pellet, and discard most of the supernatant.

1.Weigh 0.2 to 0.4g of agarose and add it to a beaker or a small reagent bottle;

2.Add approximately 10mL of PBS;

3.Melt in a microwave at high power, pausing every 5 seconds to check and stir until the agarose is completely melted;

4.Take about 200μL of melted agarose and add it to a 600μL EP tube;

5.Insert a small PCR tube into the EP tube containing the agarose to form a round, pointed bottom depression. Allow it to sit at room temperature or in a 4°C refrigerator for several minutes until the agarose solidifies. Gently twist the small PCR tube to remove it, and you will see a deep, round, pointed bottom depression formed in the agarose.

1.Gently aspirate and transfer the organoids to the bottom of a fixation mold;

2.Gently add PBS until it is higher than the edge of the agarose mold;

3.Cover and centrifuge at 1000 rpm for 5 minutes in a horizontal centrifuge;

4.Gently aspirate the PBS supernatant, leaving the organoid pellet at the bottom;

5.Add 200μL of melted agarose liquid;

6.Let it sit at room temperature until the agarose is completely solidified;

7.Cut off the bottom and lid of the fixation tube, place it in a 2 mL EP tube, fill with paraformaldehyde almost to the top, and store overnight in a 4°C refrigerator.

1.Dehydration: Remove the agarose block containing the organoid pellet, dehydrate in a graded series of ethanol, 70%, 80%, 90%, 95% ethanol for 1 hour each, and 100% ethanol for 30 minutes, until solidified.

2.Wax melting: Turn on the oven about 4 hours in advance and melt the wax at 65°C.

3.Clarification: After dehydration, transfer the agarose block to a tissue embedding box (keep it immersed in anhydrous ethanol until processed with xylene).

Note: Since xylene is toxic, perform the operation in a well-ventilated area and try to drain any residual xylene when removing it.

4.Infiltration: Immediately after clarification, transfer the block into the melted wax at 60°C for 2 hours, then change to fresh wax and infiltrate for another 2 hours.

5.Embedding: Perform the embedding on an ice box, pour the melted pure wax into the mold to one-third full, place the agarose block in the center, and then continue to pour wax until the mold is full. Once the wax block is completely solidified, carefully remove the mold.