This method can help scientists maximize productive use of pancreatic islets to assess multiple outcomes on each sample in its native tissue architecture. The main advantage of this new embedding method is that islets are evenly distributed over a well-defined area, placing many islets in the plane of section which optimizes experimental yield from this low-abundance material. Demonstrating the procedure will be Doctor Yahui Kong, a research associate in my laboratory.
To begin this procedure use a low-binding p200 pipette tip and calibrated grid under a stereo microscope to hand pick 250 islet equivalents. Transferring them into each 1.5 milliliter low-binding microfuge tube. Let the islets settle to the bottom of the microfuge tube.
Then, use a pipette with a fresh p200 tip to carefully remove most of the supernatant. Making sure to not remove any islets. Add one milliliter of PBS and centrifuge in a swinging-bucket centrifuge until the speed reaches 200 times gravity and then stop the spin.
Remove the supernatant. Repeat this entire PBS wash process for a total of two wash cycles. Next, add 500 microliters of either 10%formalin solution or 4%freshly made paraformaldehyde.
Let the sample fix at room temperature for 30 minutes. After this, use a pipette with a p200 tip to remove the fixative. Add one milliliter of PBS and centrifuge until the speed reaches 200 time gravity and then stop the spin.
Remove the supernatant and then repeat the entire PBS wash process once more for a total of two wash cycles. Prepare the desired gel in 1.5 milliliter microcentrifuge tubes as outlined in the text protocol. Next, place these microcentrifuge tubes into a heatblock at 70 degrees Celsius to warm the gel.
Use a cut micropipette tip to transfer 10 microliters of agarose blue beads per sample into a clean 1.5 milliliter microcentrifuge tube. Add one milliliter of the PBS to the beads, then centrifuge at 800 times gravity for one minute. Remove the PBS and repeat this wash once more.
After the second wash, re-suspend the beads in an appropriate amount of PBS. Centrifuge the tubes containing the islets until the speed reaches 200 times gravity and then stop the spin. Remove most of the supernatant.
Using a pair of scissors cut off the end of one 10 microliter micropipette tip for each sample. Add 10 microliters of beads to each islet tube using a clean cut off tip for each sample. Then centrifuge until the speed reaches 200 times gravity.
After this, use an uncut 200 microliter followed by a 10 microliter tip to remove as much PBS as possible. Label the microscope slides for sample identification and place them on the bench near the heatblock with the warmed gel. Using a pair of scissors, cut the ends off a few low-binding micropipette tips per sample.
Using a micropipette equipped with a cut tip, add warm gel to a tube containing islets and beads. Mix immediately while avoiding the creation of any bubbles. Apply this mixture to a slide forming a disk near the edge of the slide while leaving space the for outer gel ring.
Then, tap the slide gently a few times to settle the islets and beads. Add 20 microliters of warm gel to the sample tube and mix the new gel with any remaining islets and beads. Apply this mixture to the same slide surrounding the original disk.
Repeat as necessary using fresh pre-cut tips for each application until the disk is the desired size and thickness. Make sure to prepare each disk individually and to keep track of sample order if placing multiple disks on each side. Next, place the slides on a flat surface of wet ice and cover them.
Let the slides rest for 10 minutes or until the gel solidifes. Then, remove the slides making sure to dry the back of each one. Label a biopsy processing and embedding tissue cassette for each sample.
Use the blunt edge of a razor blade to gently push the disk from each direction to free it from the glass. When it slides easily, slowly push the disk off the slide and place the disk flat side down directly onto the paper. It can be difficult to keep the distinct disks intact while sliding it from the glass slide to the paper.
If a wrinkle occurs in the disk, allow it to return to the original position, add more gel and re-gel the slide. Fold the paper around the disk to prevent movement. Transfer this into the cassette and then close the cassette.
Submerge the cassette in a beaker filled with PBS. In this study, a modified gel disk based embedding method is used to efficiently generate a high yield of islets per section. The morphology of rodent islets is noticeably more intact after post-isolation recovery in islet medium displaying a smooth rounded surface and a compact spherical shape.
The fact that human islet morphology is similar, regardless of if it is embedded on the day of arrival or after post shipment recovery overnight, could be due to the islets recovering from isolation stress prior to shipment. In comparison, the paraffin sections obtained via the old micro tube technique, are seen to have a smaller tissue cross-sectional area with fewer islets per section and with densely packed islets and beads. As shown here, this technique is able to produce high quality islet sections for histological and immunofluorescence staining.
Insulin and glucagon staining show the diverse composition and heterogeneity of islet architecture demonstrating known architecture differences between human, mouse, and rat islets. These images also represent serial sections of the same islets. Demonstrating that this technique is capable of obtaining multiple sections from the same islets.
Once mastered, this technique saves time over the microcentrifuge technique. Forming the disk on a flat surface rather than in a tube makes it easier to physically transfer the disk to the cassette for processing. Though this method can provide insight into islet biology, it can also be applied to other cell blocks or friable tissues that are difficult to section.
While attempting this procedure, it's important to remember to use a small volume gel to concentrate tissue in a small area and form the disk on a flat surface. It is also critical to use low-binding microcentrifuge tubes and pipette tips to improve tissue yield. This technique paves the way for researchers in the field of islet biology to explore cell type composition and heterogeneity, cell-cell interaction, and gene regulation in whole cultured islets in their native architecture.
The ability to generate 10 or more sections containing many islets from a single experimental condition allows quantification of multiple outcomes on the same material, improving experimental efficiency. Applying this technique to limited abundance tissue samples may offer benefits to other fields as well. Elements of this procedure may need to be modified to meet user needs.
The fixation intensity and the division should be optimized. Thicker or larger disk can be generated using this technique. Simple processing steps for embedding may need to be nestled and they should be optimized.
After watching this video, you should have a good understanding of how to generate a high quality agarose cell block for paraffin sections of whole cultured pancreatic islets.
