The overall goal of this protocol is to demonstrate an optimized, passive optical-clearing method that is suitable for use with human pancreas tissue. This method can help answer key questions in the neurobiology field, relating to diabetes, by providing details on the innervation of islets. The main advantage of this technique, is that the clearing method ensures transparency of human pancreatic tissue for use in high-resolution, confocal imaging.
To Begin, fix a pancreas sample by placing it in freshly prepared 4%paraformaldehyde, and incubate the sample at four degrees Celsius for 48 hours. Next chill a flask by placing it in a bucket with ice. And then place the bucket on top of a magnetic stir plate.
Make sure the flask is sitting flat, and add a magnetic stir bar. Pour 147.8 milliliters of ice-cold distilled, deionized water into the flask. Then add 20 milliliters of 0.1 molar PBS, 20 milliliters of an ice-cold 40%acrylamide solution, 12.2 milliliters of a 16%paraformaldehyde solution, and 250 milligrams of VA-044 initiator.
Mix the entire hydrogel solution for at least 10 minutes. Next place a 15 milliliter conical tube in the ice next to the flask containing the hydrogel solution. Pipette 14 milliliters of monomer solution into the tube.
and add one piece of the fixed and sectioned tissue sample. Then cap the tube. Incubate the sample in the monomer solution for 3 days at four degrees Celsius, while protected from light.
Following the incubation, place the sample on ice. Next connect tubing to a nitrogen tank through a stopcock. While keeping the sample on ice, carefully use an 18 gauge hypodermic needle to pierce the cap of a conical tube containing the sample on one side.
Insert the needle into the tube until it is under the surface of the liquid monomer solution. Then use another 18 gauge hypodermic needle to puncture the opposite side of the cap, but do not allow it to become submerged in the solution, so that it may act as a vent. Connect the tubing from the nitrogen tank to the hypodermic needle submerged beneath the hydrogel, and slowly turn on the nitrogen, until it is bubbling steadily through the liquid.
Once deoxygenated, quickly remove both needles, and cover the cap with a paraffin film, to prevent any further exchange of gases between the tube and the environment. Finally, place the sample in an incubator at 37 degrees Celsius, for three hours, to polymerize the hydrogel. After polymerization, pour away any of the remaining hydrogel.
Then, wash the sample in 3 to 5 exchanges of 0.01 molar PBS for 15 minutes each wash step. Once washed, transfer the sample into a 50 milliliter conical tube containing 40 milliliters or clearing buffer. Incubate the sample in the clearing buffer at 37 degrees Celsius and change the sample to fresh clearing buffer every other day.
To check for proper clearing, hold the sample up to a light and insure that the sample allows light through it but retains some tan coloring in the exocrine regions. An over cleared sample will appear frayed at the edges and the texture will be very soft when picked up with forceps. It is common for a sample to clear unevenly.
Exchange the clearing buffer with 40 milliliters 0.01 molar PBS, and place the samples on a shaker at 60 RPMs for one day at room temperature. Perform four to five buffer changes and let the final wash continue over night. Next, prepare PACT staining buffer in a 2 milliliter flat bottom tube and add 2%normal serum to 1 milliliter of the base PACT buffer.
Then, add approximately five times the standard amount of primary antibody to the staining buffer. Using a spatula, remove the sample from the wash buffer and dab the excess buffer onto a paper towel. Place the sample into the tube with the primary antibody solution, and incubate it in the solution for two to four days at room temperature, on a shaker, at 60 RPM.
Following the incubation, remove the antibody solution and add 0.01 molar PBS. Wash the samples thoroughly on the shaker at 60 RPM, changing to fresh buffer four to five times and leaving the final wash on the shaker overnight. Next, add the secondary antibody at 1 to 200 in the PACT staining buffer with 2%added serum.
Use a spatula to remove the sample from wash buffer and dab the excess buffer off, onto a paper towel. Then place the sample in the tube with the secondary antibody solution. Protect the sample from light while incubating the sample at room temperature on a shaker at 60 RPMs for two days.
Following the incubation, remove the antibody solution and replace it with 0.01 molar PBS. Wash the samples thoroughly on the shaker at 60 RPM, changing to fresh buffer four to five times, and leaving the final wash on the shaker overnight. To begin, prepare the refractive index matched solution buffer by first weighing out 11 grams of non-ionic density gradient medium and carefully transferring it to a 50 milliliter conical tube.
Then add 5 milliliters of 0.02 molar phosphate buffer using a spatula to release air from the powdered non-ionic density gradient medium. If necessary, bring the volume up to 10 milliliters, using more of the 0.02 molar phosphate buffer, mix it with a spatula, and then scrape the excess off the spatula into the tube. Incubate the buffer at 37 degrees Celsius until it fully dissolves, inverting and gently mixing it periodically.
Transfer samples into the refractive index match solution buffer and incubate them on the bench protected from light for two to four days before imaging. Just before imaging, place a small amount of the refractive index match solution into an eight well coverslip bottom chamber slide. Then add the sample to the well and cap the slide.
In the human pancreas, islets can be delineated by Insulin, Glucagon, and Secreted Grana Three for beta cells, alpha cell, or all endocrine cells respectively. Schwann cells appear white, and endocrine cells are shown stained by either Insulin or Glucagon. Nerves, coursing next to blood vessels at the islet periphery stand out as white lines and extend into the islets.
Here, contact between the Schwann cells and endocrine cells can clearly be seen. Here, a sample prepared using the methods shown in this video was stained for vasoactive intestinal peptide and imaged using a light sheet microscope. The nerve fibers are clearly seen in high resolution and are shown wrapping a duct in the foreground and a ganglion in the background.
While attempting this procedure, it's important to remember to use pancreas acinar regions that are free of major ducts and vessels.
