Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

In combination with live cell calcium imaging, the acute pancreas tissue slice technique enables the study of intercellular waves and multicellular functional connectivity with a high resolution over long time periods. The main advantages of this technique are that it is fast, preserves tissue architecture, and reduces limited enzymatic and mechanical stress while maintaining a high yield of tissue. By detecting functional and morphological changes during disease progression, this technique helps our understanding of the pancreas related diseases, such as diabetes.

This tissue slice technique can be applied to brain, pituitary, and adrenal tissues with an emphasis on the preservation of intercellular contact, paracrine interactions, and tissue architecture. To prepare for the injection of agarose into the mouse pancreas, fill a five milliliter syringe with 40 degrees Celsius liquid agarose and equip the syringe with a 30 gauge needle. After carefully covering the needle with a cap, place the syringe needle end down, with the entire volume of agarose below the water surface, in a 40 degrees Celsius water bath.

Bubble a bottle of extracellular solution with carbogen, continuously on ice, at 1.5 milliliters per minute at barometric pressure and room temperature to ensure oxygenation and a pH of 7.4. To perform the agarose injection place a euthanized adult mouse under a stereo microscope and access the mouse abdomen via laparotomy. Gently move the gut to the left side to expose the common bile duct and use forceps to slightly lift the duodenal end of the duct to locate the papilla of Vater.

Use a hemostat to clamp the bile duct at the duodenal papilla to prevent leakage of the agarose from the duct into the duodenum and use a small sharp forceps to reach under the common bile duct to break the membrane that attaches the duct to the pancreatic tissue. After clearing as much fat and connective tissue from the duct as possible, place the duct perpendicularly onto the tips of a pair of larger forceps, and pressing firmly on the syringe plunger, inject the viscous liquid agarose into the proximal end of the common bile duct for 20 to 30 seconds. When the tissue becomes whitish and slightly distended remove the syringe and slowly pour 20 milliliters of the bubbled ice-cold extracellular solution onto the pancreas to cool the tissue and to harden the agarose.

To acquire pancreatic tissue slices use forceps and fine tough cut scissors to gently transfer the agarose injected pancreas into a 100 millimeter Petri dish containing 40 milliliters of ice-cold extracellular solution. Swirl the dish to rinse the tissue and transfer the pancreas into a second dish of ice-cold extracellular solution. Use the forceps and the scissors to cut the whitish, well-injected pancreas section into up to six 0.1 to 0.2 cubic centimeter pieces and remove any remaining connective and fatty tissue from the tissue pieces.

Place the cleaned blocks into a 35 millimeter non-sticky bottom Petri dish containing approximately five milliliters of 40 degrees Celsius liquid agarose and immediately place the Petri dish on ice. When the agarose has hardened, hold the Petri dish upside down over the lid of a 100 millimeter Petri dish and use one half of a razor blade to carefully cut into the margin between the lateral wall of the Petri dish and the agarose to remove the agarose from the dish. Use the razor blade to cut individual cubes, each containing one tissue block of agarose, from the freed agarose and use cyanoacrylate glue to attach the blocks to the sample plate of a vibratome.

Fill the cutting chamber of the vibratome with 150 milliliters of ice-cold extracellular solution continuously bubbled with carbogen. Screw fix the sample plate onto the vibratome and mount a new razor blade. Surround the chamber with ice and add two 10 milliliter volume ice cubes made with extracellular solution supplemented with six millimolar glucose.

Set the slicer to 0.05 to 1 millimeter per second and 70 hertz, then begin slicing to acquire 140 micron thick agarose slices with a 20 to 100 square millimeter surface area. Use a fine paint brush to carefully transfer each slice into a 100 millimeter Petri dish filled with 40 millimeters of HEPES buffer supplemented with six millimolar glucose. For calcium dye preparation, dissolve 50 micrograms of cell permeable calcium indicator dye, 7.5 microliters of DMSO, and 2.5 microliters of the poloxamer, and 6.667 milliliters of HBS in a 15 milliliter screw cap tube.

Use a pipette to thoroughly mix the solution for 20 seconds before submerging the tube in an ultrasonic bath chamber and vortexing, each for 30 seconds. Then aliquot 3.333 milliliters of the resulting calcium indicator dye solution into one 5 milliliter Petri dish per 10 slices of tissue to be labeled. For dye loading, use a thin soft paintbrush to transfer up to 10 tissue slices to each dish of dye solution and place the dishes onto an orbital shaker for 50 minutes at room temperature and 40 revolutions per minute protected from light.

At the end of the incubation, transfer up to 20 stained slices into individual 60 milliliter Petri dishes filled with dye-free HBS. For calcium imaging by confocal microscopy select a 20 or 25 times magnification and set the acquisition mode to time-lapse imaging, the pinhole to 100 to 200 microns, and the excitation and emission for the fluorophore used in the experiment. Mount the recording chamber and perfusion system onto the temperature controlled stage of the microscope and position the inlet and outlet on the far edges of the recording chamber.

Set the inflow and outflow rates to one to two milliliters per minute. Set the temperature of the profusion system to 37 degrees Celsius and initiate the profusion with the non-stimulatory solution. To record the calcium dynamics of the tissue place a single tissue slice into the recording chamber and immobilize the tissue with a U-shaped platinum weight with a taut nylon mesh.

Use the bright-field option to locate the structure of interest and use live imaging to position the studied structures into the field of view. To optimize the signal-to-noise ratio, adjust the laser power, detector amplification, and line averaging binning while keeping the laser power minimal. Adjust the focal plane to 15 microns below the cut surface to avoid recording from potentially damaged cells and acquire the images.

Setting the sampling frequency to one to two hertz to allow the initial detection of individual oscillations and recording a high resolution image. If available, use an online chart to obtain instant feedback on the preparation response, over illumination, photobleaching, and mechanical drift. When all of the images have been acquired, save the data for later analysis.

Here an example of an optimal tissue slice can be observed in which the cell permeable calcium indicator dye was successfully loaded into the pancreatic tissue slice. In contrast, these tissue slices are not optimal for imaging due to either an unsuccessful dye penetration, a lack of islet cells, or an abundance of necrotic tissue on the samples surface. High resolution images of a pancreatic tissue slice can be used to delineate distinct regions of the pancreatic tissue, such as the islets of Langerhans, acinar tissue, or the pancreatic duct.

Stimuli can be used to functionally discriminate between different islet cells or between islet and non-islet cells. For example, beta cells typically respond to a square pulse glucose stimulation by exhibiting a transient increase in intracellular calcium followed by a fast calcium oscillations on a sustained plateau. In contrast, non-beta cells respond with faster and more irregular oscillations.

A delay in the onset of intracellular calcium increase after stimulation, as well as the heterogeneity and delays among individual cells can also be measured. The same parameters can be used to describe the deactivation phase. Here a schematic presentation of the intracellular calcium oscillation duration, frequency, and percentage of active time can be observed.

When imaging at acquisition rates greater than 10 hertz, calcium waves that repeatedly spread across the islet can be clearly recognized. Clamp the common bile duct as closely to the duodenum as possible to prevent accidentally occluding the branch which is entering the pancreas. Also, do not stop depressing the plunger during the injection because the agarose will immediately harden in the needle.

The acute mouse pancreatic tissue slice technique may also be used with the patch clamp method to study ion channel currents and access cytosis as well as immunohistochemistry studies and secretary studies.