Our protocol will help answer the impact that immune cell interactions have on islet functionality. The live pancreatic tissue slice method allows for islet physiology and function to be studied while maintaining the tissues underlying pathologies and native microenvironment. This method could provide insight into the disease processes of pancreatic illnesses, such as pancreatitis, type I diabetes, and type II diabetes.
To begin, arrange the blocks on the specimen holder in such a way that they do not exceed blade width, ensuring that the blade can move forward the least possible distance when the vibratome moves slowly. Apply a line of super glue on the specimen holder, making a thin layer using the end of the glue dispenser, then flip the tissue blocks onto the glue so that the side close to the tissue faces upward. Gently push down the blocks and let the glue dry for three minutes.
Next, attach the plate to the vibratome with a screw and adjust the blade height and distance being traveled so that the blade moves over the length of blocks and just barely above them. Gently nudge the blocks with the forceps to check that the glue has dried, and then fill the vibratome tray with a chilled extracellular solution until the blade is covered. Set the vibratome to make slices of 120 micrometer thickness and start it, then observe the slices coming off the tissue blocks.
Lift the slices when they float off the block by placing a paintbrush or forceps below them and place the slices in Krebs-Ringer bicarbonate buffer containing three millimolar D-glucose and trypsin inhibitor. Place the plates containing the slices on a rocker and incubate at room temperature for an hour at 25 RPM. If the slices need to be kept for longer, place the slices in 15 milliliters of slice culture medium and place the plate in an incubator.
Incubate the prepared slices at 37 degree Celsius for same day studies or transfer the slices that are cultured at 24 degree Celsius overnight to 37 degree Celsius for at least one hour before the experiment. Switch on the microscope at least one hour before the recording and equilibrate the stage top incubator to 37 degree Celsius. Secure the cover glass bottom Petri dish containing the slice on the stage.
Focus the microscope by setting the 10X objective in the brightfield mode and locate the islets using the bright field mode by looking for orange brown colored ovals within the slice. Switch the microscope to confocal imaging by pressing the CS button on the microscope's touch screen controller. To view the islets using reflectance, turn on the 638 laser detector and PMT detector.
Set the laser power between one and 2%and turn off the notch filters. Use the 405 nanometer laser and PMT detector to observe the nucleic acid stain. The hybrid detector for the CD8 antibody detection and the 488 nanometers laser and hybrid detector to view the insulin tetramer.
Center the islet of interest in the field of view using the X and Y knobs of the stage controller. Once an islet of interest is located, switch to the 20X objective and zoom in so the islet takes up most of the frame. Take a z-stack of the islet, then find the best optical sections where to the cells are alive and any surrounding immune cells are in focus.
Set the microscope to record in XYZT mode and optimize the settings to record a z-stack of the selected steps every 20 minutes over a period of several hours. The islets in a pancreatic tissue slice were visualized using brightfield and reflected light microscopy. The insulin in the islets increases the granularity and causes absorption of large amounts of reflected light, increasing the visibility of the islets.
The viability of a live human pancreatic tissue slice was assessed by staining. The live cells are indicated in green and the dead cells in blue. Another positive indicator for viability is observable basal activity.
When a live pancreatic slice was loaded with a cell permeable calcium indicator, glucose stimulation was observed in both mouse and human pancreatic slices. The fluorescence of individual cells during glucose stimulation was also quantified. Intact and diseased islets were observed using dithizone staining and reflected light microscopy.
The diseased islets begin to lose granularity due to immune cell infiltration and cell death. Immune cell populations can be identified using CD8 antibodies and insulin tetramer staining. Co-staining indicates that the cells are effector T-cell that are specifically targeting the insulin antigen.
The most critical thing to remember in this procedure is to keep the slices in solutions protease inhibitor at all times. Following this procedure, numerous functionality in immune cell experiments can be performed allowing for the effect of immune cell interactions and islet function to be studied.
