TITLE: Human Pancreas Processing for Multiple Applications
AUTHORS: Diane Saunders, Rita Bottino, Marcela Brissova, Angela Kruse, Jamie Allen, Carrie  Romer, Danielle Gutierrez, Jeff Spraggins, Alvin Powers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Procuring and preparing human pancreas for paraffin-embedding, fixed cryosections, flash frozen sections, and RNA isolation.</div><br/><div class = "text-block">This protocol was developed in collaboration with Dr. Rita Bottino of Imagine Pharma and Allegheny Health Network and is based on protocols developed by Dr. Marcela Brissova of Vanderbilt University. </div></div>

[STEPS]
?. [Preparing Pancreatic Cross-Sections]
Upon arrival, transfer the pancreas and transport solution (UW or HTK) into a dissection tray placed on ice. Transfer enough of the transport solution to keep the pancreas submerged.
?. [Preparing Pancreatic Cross-Sections]
Dissect the pancreas by carefully removing duodenum, mesentery, fat, and spleen.
If there are pieces of duodenum and/or spleen, please retain and freeze as tissue blocks *see Human Pancreas Processing for Flash Frozen Sections protocol below
?. [Preparing Pancreatic Cross-Sections]
Once the pancreas is cleaned, blot with a sterile sponge (Covidien #8044) and quickly take its weight by placing it into a clean plastic weigh boat.
?. [Preparing Pancreatic Cross-Sections]
Immediately transfer the pancreas on an ice-cooled dissecting platform and take a photograph with a ruler as shown in Figure 1.
?. [Preparing Pancreatic Cross-Sections]
Apply marking dyes (Cancer Diagnostics, Inc. #MD2000) to length of organ as follows:o Anterior: blueo Superior: blacko Posterior: greeno Inferior: red
?. [Map of Pancreatic Slices]
Once the pancreas has been marked, start collecting approximately 5-mm thick cross-sectional slices starting from the Head and moving laterally towards the Tail as outlined in Figure 2. Each cross-section should contain dye markings on the exterior to provide spatial orientation.
?. [Map of Pancreatic Slices]
Label cross-sections following the naming nomenclature and controlled vocabularies developed for Vanderbilt LIMS, which keeps track of the position of each cross-section in a given anatomical location and processing procedure.
?. [Map of Pancreatic Slices]
When dividing each tissue cross-section into quadrants, orient it medial side down and attempt to bisect dye markings so that each tissue piece contains 2 dye colors (Figure 3).
?. [Human Pancreas Processing for Paraffin-embedding (PE)]
Cut each tissue cross-section into four quadrants (A, B, C, D; see Figure 3) and place all four tissue pieces into the same 50-mL Falcon tube containing   of freshly prepared fixative (4% PFA/100 mM PBS). Fix  , at least  , at 4ºC under mild agitation using an adjustable tilt rocker (LabNet).
40 mL
?. [Human Pancreas Processing for Paraffin-embedding (PE)]
Wash tissue four times in   of 100 mM PBS at 4ºC for a period of 2-3 hours, rocking. Blot the tube with paper towel before adding the fresh washing solution.
40 mL
?. [Human Pancreas Processing for Paraffin-embedding (PE)]
After the last wash, add    of 70% ethanol and keep at 4ºC.
40 mL
?. [Human Pancreas Processing for Paraffin-embedding (PE)]
Ship tissue in sealed tubes on ice packs.
?. [Human Pancreas Processing for Fixed Cryosections (PCMC)]
Cut each tissue cross-section into four quadrants (A, B, C, D; see Figure 3) and place all four tissue pieces into the same 50-mL Falcon tube containing   of freshly prepared fixative (4% PFA/100 mM PBS). Fix for   on ice under mild agitation using an adjustable tilt rocker (LabNet).
40 mL
?. [Human Pancreas Processing for Fixed Cryosections (PCMC)]
Wash tissue four times in  of 100 mM PBS on ice for a period of 2-3 hours, rocking. Blot the tube with paper towel before adding the fresh washing solution.
40 mL
?. [Human Pancreas Processing for Fixed Cryosections (PCMC)]
After the last wash, equilibrate the tissue in    of 30% sucrose/10 mM PBS at 4ºC overnight. Tissue will settle to bottom of the tube.
40 mL
If the pancreas is not cleaned sufficiently from fat, it will never drop to the bottom.
?. [Human Pancreas Processing for Fixed Cryosections (PCMC)]
Prepare a pre-labeled cryomold (VWR #25608-916) and fill it halfway with CMC compound. Pour the pancreas with some sucrose into a 10-cm petri dish. Pick the pancreas with a pair of fine forceps and blot it with Kimwipes to remove an excess of sucrose. Place the tissue into the CMC-containing cryomold. Using forceps, push the tissue lightly to bottom of the cryomold, medial side down. Add more CMC to completely fill the cryomold.
?. [Human Pancreas Processing for Fixed Cryosections (PCMC)]
Freeze the tissue on a dry ice block. As CMC starts freezing, push the tissue again lightly to bottom of the cryomold.
?. [Human Pancreas Processing for Fixed Cryosections (PCMC)]
When CMC compound is frozen, wrap the cryomold containing the tissue in a pre-labeled aluminum foil, place it a storage bag, seal, and store in a storage box at -80ºC.
Aluminum foil and zip-lock bag prevent the block from drying.
?. [Human Pancreas Processing for Fixed Cryosections (PCMC)]
Ship tissue blocks on dry ice.
?. [Human Pancreas Processing for Flash Frozen Sections (FCMC, FF)]
Cut each tissue cross-section into four quadrants (A, B, C, D; see Figure 3) and place into metal weigh boat with sample on top of slurry. Cover box with lid and allow tissue to completely freeze.
?. [Human Pancreas Processing for Flash Frozen Sections (FCMC, FF)]
For FF-CMC, label edges of the molds and place embedding mold with CMC on top of the slurry. Add tissue, medial side down, when the CMC starts to become opaque. Slowly add more CMC on top of the tissue until it is covered, keeping the lid closed over the slurry as much as possible. When completely frozen, wrap mold in foil.
?. [Human Pancreas Processing for Flash Frozen Sections (FCMC, FF)]
FF-RNA, label pieces of aluminum foil and corresponding storage bags. Place one tissue piece onto each piece of foil and wrap tightly.
?. [Human Pancreas Processing for Flash Frozen Sections (FCMC, FF)]
Place all samples in labeled storage bags and store at -80ºC.
?. [Human Pancreas Processing for Flash Frozen Sections (FCMC, FF)]
Ship tissue blocks on dry ice.
?. [Human Pancreas Processing for RNA Isolation (RNA)]
Cut each tissue cross-section into four quadrants (A, B, C, D; see Figure 3) and place all four tissue pieces into a tared plastic weigh boat. Transfer to 15 mL Falcon tube with approximately 5 volumes of RNAlater (e.g., a 1 g sample requires about  of solution). Keep at 4ºC.
5 mL
?. [Human Pancreas Processing for RNA Isolation (RNA)]
Ship tissue in sealed tubes on ice packs.