The overall goal of this procedure is to dissect the human pancreas in a manner that maintains the anatomical orientation. This is accomplished by removing the duodenum and spleen from the pancreas, then clearing away excess fat and vessels from the pancreas. The second step is to paint the anterior surface with blue ink and posterior surface with black ink.
Next, the pancreas is divided into three regions. Each region is then sliced crosswise into even slabs and placed onto paraffin cassettes, OCT molds or minced further and placed into vials. Ultimately, samples are collected from all three regions of the pancreas, while the correct anatomical orientation is maintained for future analysis.
This method can help address key questions in type one diabetes research, such as the mechanisms underlying loss of beta cell mass, or function, and the nature of immune cell attack. Visual demonstration of the pancreas processing method is critical since maintaining the in vivo orientation of the pancreas requires attention to detail. Demonstrating the procedure will be Linda Snyder, histo technologist and Emily Montgomery, biological scientist in my laboratory.
Begin by preparing all of the materials needed for the procedure. First, label the cassettes that will hold the paraffin blocks with a donor identifier, sample type, aliquot number, and any additional information. Next, label the OCT molds in the same manner.
Cut enough pieces of aluminum foil to wrap each OCT block and set them aside for use later. Prepare all of the vials needed to store the fresh and frozen samples. Add RNA later and RPMI media when appropriate.
Next, prepare the work areas, place dissection boards, sterilized instruments, and all other supplies needed for the procedure in both a biosafety hood and on a countertop. Have all cassettes and OCT molds arranged in order of use.Nearby. Create a freezing bath by adding enough isop pentane to cover the dry ice in the bottom of a styrofoam box.
To begin the dissection, remove the duodenum and spleen. Start separating the duodenum and pancreas at one end, and continue until completely detached. The spleen is loosely attached and is easily removed from the fat and pancreas.
Next, clean the pancreas of extraneous fat vessels and nerves using a combination of blunt and sharp dissection. Set aside the fat and lymph nodes to be processed later. Once cleaned, place the pancreas on a dissecting board covered with paper towels.
Maintaining the correct orientation of the pancreas is the single most difficult aspect of this procedure. To ensure success, the anterior and posterior surfaces are painted with different colored inks. Dip a cotton tipped applicator in blue ink, spread the ink on the anterior surface of the pancreas and blot off the excess.
Next, dip a new applicator in black ink and spread it on the posterior surface of the pancreas. Return the pancreas to its normal orientation. With the anterior surface facing up, the pancreas is now ready to be cut into three sections.
First, cut the junction between the head and body. Next, divide the remaining portion into approximately equal portions to separate the body and tail, record the weight of each region of the pancreas. Starting with the head section.
Divide each region into approximately 0.5 centimeter sections by cutting in a transverse manner, similar to cutting a loaf of bread. Once each region of the pancreas has been sliced, remove the last section of the head region, which is the head body junction section, and place it on a separate dissecting board. Mince this section into small pieces and divide the pieces evenly between labeled cryo vials with and without RNA.
Later, the vials are then frozen. Following standard procedures. Repeat this procedure for the body tail junction section.
Once the minced samples have been collected, lean all transverse sections to the right. Alternating sections of each region will be removed. For paraffin and OCT blocks, remove the first section for paraffin blocking.
If a section is too large, cut it in half. Label the cassettes as A and B.If the piece is still too large, cut each section perpendicular to the previous cut, and then label the cassettes A through D in a clockwise manner. As seen here, position the cassette so that the label is to the left.
Next, place the tissue into the cassette and close the lid. Transfer the cassette to a container containing neutral buffered formin. Number the block sequentially within each region, starting with the most medial section for OCT blocks.
Pour a thin layer of OCT media into a pre-labeled mold Before introducing the tissue, cover the tissue with OCT media and rapidly freeze the sample for later use returning to the tissue that was set aside earlier. Isolate the pancreatic lymph nodes by trimming away the fat and connective tissue surrounding the node capsule. Place the nodes in a sterile dish containing DMEF mince large nodes into pieces no larger than 0.5 centimeters.
Once the pancreatic lymph nodes are isolated, divide the tissue between the prepared vials and cassettes for further processing. After finishing the lymph nodes, the spleen is minced and stored in a similar manner. To process the duodenum, use a moistened gauze pad to wipe the mucosa clean of mucus, snip off the mucosa, and then cut it into sections.
Divide the mucosa samples as shown earlier. Using this procedure, representative samples of each region of the pancreas are collected as seen here. The weights of the head, body, and tail region were approximately equal.
After watching this video, you should have a good understanding of how to process a human pancreas into different sample types, specifically the ability to maintain the orientation, as well as how to isolate lymph nodes from the peripancreatic fat.
