Methods
1.1 Harvest and Fix Spheroids
The following steps are used to harvest and fix spheroids:
1. Place a pipette tip near the bottom of the well of a spheroid-containing 96-well plate and aspirate both the medium supernatant and spheroids. Be careful to avoid touching the tip to the bottom of the well to prevent crushing the spheroids in the bottom.
2. Pipette the spheroids into a 1.5 mL microcentrifuge tube.
3. Allow gravity to settle the spheroids to the bottom of the tube. Alternately, centrifuge the tube for 10 s at 200 × relative centrifugal force (RCF).
4. Aspirate the supernatant from the tube.
5. Use 500 μL of 4% PFA (Paraformaldehyde) to fix the spheroids. Spheroids should be fixed at room temperature for at least 1 h or
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Remove the fixative.
7. Wash the tissues two times with phosphate-buffered saline. Discard the supernatant.
8. Stain the spheroids with eosin, if desired. An eosin stain will make it much easier to see and handle during the rest of the procedure.
1.2 Create a Plug of Spheroid-Containing Agarose Gel
After the spheroids have been fixed and stained, follow these steps to create a plug of Agarose Gel:
1. Heat the Agarose gel in a 65 °C water-bath to melt the agarose. After it melted, maintain its temperature at 55 °C until it is ready for use.
2. Transfer the spheroids from the 96-well plate to a 15 mL centrifugal tube using a 1000 μL pipette
3. centrifuge the tube for 5 min at 1000 rpm to form a pellet.
4. Remove the supernatant and transfer the pellet to a 1.5 mL microcentrifuge tube.
5. Suspend the pellet in eosin to make the spheroids more visible.
6. centrifuge the tube
7. remove excess eosin with a
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Remove the processed spheroid-containing plug from the cassette. 1.4 Embed the Processed Spheroid-Containing Plug for Sectioning
Follow these steps to embed the spheroid-containing plug in wax for sectioning:
1. Remove the spheroid-containing plug from the processing cassette. Using the paraffin-embedding station, fill the depressed portion of a metal mold with liquid paraffin and, with the spheroids facing the bottom of the depressed portion of the mold, place the spheroid-containing plug in the paraffin wax.
2. Separate the lid from the base of the cassette and adhere the base of the cassette to the metal mold. The cassette base enables attachment of the spheroid-containing wax block to the microtome.
3. Allow the wax in the cassette to solidify on the cold plate, and then place it in ice for 10 min until the wax is fully solidified. Note (you can store it in -4 OC until it processed)
4. Remove the cassette from the ice and separate the plastic cassette containing the wax and spheroids from the metal mold.
1.5 Section the Embedded Spheroid-Containing Plug and Create a Slide
After the spheroid-containing plug has been embedded in paraffin, follow these