Feb 03, 2026
Human Pancreas Processing
- Anastasia Coldren1
- 1Vanderbilt University Medical Center
- Powers Brissova Lab
Protocol Citation: Anastasia Coldren 2026. Human Pancreas Processing. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9oqwmv3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 16, 2025
Last Modified: February 03, 2026
Protocol Integer ID: 220346
Abstract
Human Pancreas Processing
Guidelines
Human sample collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee.
Materials
*Preparing Pancreatic Slices*:
Supplies:
Sterile Sponge (Covidien, 8044)
Plastic Weigh Boat
Ruler with cm
Microsurgical tools
Equipment:
Dissection Tray
Analytical Balance
Ice-cooled Dissecting Platform
Digital camera
*Human Pancreas Processing for Paraffin-embedding (PE)*
Reagents:
Sodium Phosphate dibasic anhydrous (Na2HPO4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5011
KH2PO4Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5655
NaClFisher ScientificCatalog #S640-10
KClMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
70% ethanolVWR International (Avantor)
Supplies:
50 mL conical tubes
Fine scissors
10 mL Petri dishes
Equipment:
Equipment
Adjustable Tilt Rocker
NAME
LabNet
BRAND
S2035-DE
SKU
*Human Pancreas Processing for Flash Frozen Sections (FF)*:
Supplies:
50 mL conical tubes
Forceps
Aluminum foil
Zip-lock bags
Dry ice
Reagents:
Liquid Nitrogen
Dry ice
*Human Pancreas Processing for Fixed Cryosections (OCT)*:
Supplies:
50 mL conical tubes
CryomoldVWR International (Avantor)Catalog #25608-916
Andwin Scientific Tissue-Tek™ CRYO-OCT CompoundFisher ScientificCatalog #14-373-65
10 cm Petri dish
Fine forceps
Kimwipes
Aluminum foil
Zip-lock bags
Reagents:
PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
16% Paraformaldehyde (PFA)Fisher ScientificCatalog #50-980-487
Equipment:
Equipment
Adjustable Tilt Rocker
NAME
LabNet
BRAND
S2035-DE
SKU
*Human Pancreas Processing for Clarity (CLR)*:
Supplies:
50 mL conical tubes
Reagents:
PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
Equipment:
Equipment
Adjustable Tilt Rocker
NAME
LabNet
BRAND
S2035-DE
SKU
*Human Pancreas Processing for Transmission Electron Microscopy (EM)*:
Supplies:
50 mL conical tubes
Reagents:
PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
8% GlutaraldehydeElectron Microscopy SciencesCatalog #16020
Sodium Cacodylate BufferElectron Microscopy SciencesCatalog #11652
Equipment:
Equipment
Adjustable Tilt Rocker
NAME
LabNet
BRAND
S2035-DE
SKU
Protocol materials
100 mM PBSElectron Microscopy SciencesCatalog #19344-10
70% ethanolVWR International (Avantor)
Liquid Nitrogen
Dry ice
16% Paraformaldehyde (PFA)Fisher ScientificCatalog #50-980-487
D-SucroseFisher ScientificCatalog #BP220-1
Andwin Scientific Tissue-Tek™ CRYO-OCT CompoundFisher ScientificCatalog #14-373-65
CryomoldVWR International (Avantor)Catalog #25608-916
PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144
8% GlutaraldehydeElectron Microscopy SciencesCatalog #16020
PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710
Sodium Cacodylate BufferElectron Microscopy SciencesCatalog #11652
KH2PO4Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5655
NaClFisher ScientificCatalog #S640-10
KClMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Sodium Phosphate dibasic anhydrous (Na2HPO4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5011
Troubleshooting
Before start
Prepare all reagents needed before starting this protocol.
Preparing Pancreatic Slices
Upon arrival, transfer the pancreas and transport solution (UW or HTK) into a dissection tray placed on ice. Transfer enough of the the transport solution to keep pancreas submerged.
Dissect the pancreas by carefully removing duodenum, mesentery, fat, and spleen.
Once the pancreas is clean, blot it with a sterile sponge, and quickly take its wet weight by placing it into a pre-weighed clean plastic boat.
Range of pancreatic weights
collected from neonatal and juvenile
donors in the first 10 years of life.
Transfer immediately to an ice-cooled dissecting platform and take a photograph with a ruler as shown below.
Example of pancreatic tissues from
young donors of different age categories.
Immediately after photographing, start collecting approximately 2 mm thick slices as outlined below. Collect slices in head-to-toe order as shown below.
Map of pancreatic slices with reference to their anatomical location and processing procedure.
The number of slices and their processing will be determined on a case-by-case basis depending on tissue availability, age category, and requirements for downstream analyses.
Label pancreatic slices following the naming nomenclature and controlled vocabularies developed for Vanderbilt LIMS, which includes the position of each slice in a given anatomical location and the processing procedure.
LABELING EXAMPLES for a 10-year-old male donor without diabetes:
Slices collected from the pancreatic head:
- UNOS ID-ND-10y-M-PANC-OCT-H1
- UNOS ID-ND-10y-M-PANC-PE-H2
- UNOS ID-ND-10y-M-PANC-EM-H3
- UNOS ID-ND-10y-M-PANC-FF-H4
If the next set of slices is also collected from the pancreatic head:
- UNOS ID-ND-10y-M-PANC-OCT-H5
- UNOS ID-ND-10y-M-PANC-PE-H6
- UNOS ID-ND-10y-M-PANC-EM-H7
- UNOS ID-ND-10y-M-PANC-FF-H8
LABELING EXAMPLES for a male donor less than 1 year of age. Each slice would be divided into quadrants and labeled as follows:
- UNOS ID-ND-6mo-M-PANC-OCT-H1A
- UNOS ID-ND-6mo-M-PANC-PE-H1B
- UNOS ID-ND-6mo-M-PANC-EM-H1C
- UNOS ID-ND-6mo-M-PANC-FF-H1D
LIVING SLICES: Prior to pancreas processing, tissue from the Tail region will be removed to generate 100-140 living pancreatic slices following existing nPOD protocol.
Human Pancreas Processing for Paraffin-embedding (PE)
20h
Prepare 1 L 100 millimolar (mM) PBS:
Note
Steps that use this recipe:
12.07 g Sodium Phosphate dibasic anhydrous (Na2HPO4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5011
2.04 g KH2PO4Merck MilliporeSigma (Sigma-Aldrich)Catalog #P5655
8.0 g NaClFisher ScientificCatalog #S640-10
2.0 g KClMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Adjust to 7.5 .
Filter through a 0.22 µm filter.
Note
100 millimolar (mM) PBS can be stored at 4 °C for up to 1 month.
Prepare 40 mL fixative for each slice: 4% PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710 in prepared 100 millimolar (mM) PBS
Transfer contents of 10 mL vial of PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710 into a 50 mL conical tube.
Add 30 mL 100 millimolar (mM) PBS. Mix and place the tube On ice .
Prepare and label one 50 mL conical tube with 40 mL of freshly-prepared fixative for each slice.
Cut each cross-sectional slice from the Head, Body, and Tail into four quadrants (A, B, C, D) and place all 4 tissue slices into the same 50 mL tube with fixative.
Note
For donors less than 1 year of age, keep the entire slice intact.
Place tube onto an adjustable tilt rocker at 4 °C , and set to mild agitation. Allow tissue to fix at least 18 hours.
18h
Decant the fixative and blot the tube onto a paper towel. Add 40 mL 100 mM PBSElectron Microscopy SciencesCatalog #19344-10 and place back onto the rocker. Wash for 00:30:00 .
30m
Repeat STEP 11 3 times for another 01:30:00 .
1h 30m
After the final wash, add 40 mL 70% ethanolVWR International (Avantor) . Place tubes at 4 °C .
Ship tissue tubes in sealed tubes to Vanderbilt University Medical Center with ice packs.
Human Pancreas Processing for Flash Frozen Sections (FF)
Fill one 50 mL conical tube with 2-5 mL Liquid Nitrogen for every tissue slice to be procured and place on Dry ice .
Cut a piece of aluminum foil (about 5 square inches ) for each tissue slice, label, and place on Dry ice to cool down.
Collect about2 mm thick cross-sectional slices from the Head, Body, and Tail (see figure in Step 5 ).
Cut each slice into 4 quadrants (A, B, C, D), and drop each piece of tissue, one at a time, into the same tube.
Note
For donors less than 1 year of age, keep the entire slice intact and drop into the tube.
Allow the Liquid Nitrogen to evaporate on Dry ice .
Using forceps, transfer the frozen tissue to a pre-labeled and pre-cooled piece of aluminum foil. Wrap the tissue in the foil, and place in a zip-lock bag. Seal and store at -80 °C .
Note
Aluminum foil and zip-lock bag keep the tissue from drying out.
Ship tissue blocks to Vanderbilt University Medical Center on Dry ice .
Human Pancreas Processing for Fixed Cryosections (OCT)
9h
Label a CryomoldVWR International (Avantor)Catalog #25608-916 and a piece of foil (about 5 square inches ) for each tissue.
Prepare 100 millimolar (mM) PBS per .
Prepare 4% PFA solution:
Transfer contents of 10 mL16% Paraformaldehyde (PFA)Fisher ScientificCatalog #50-980-487 vial to a 50 mL conical tube.
Add 30 mL prepared 100 millimolar (mM) PBS.
Mix and place the tube On ice .
Prepare 30% sucrose solution:
Weigh out 15 g D-SucroseFisher ScientificCatalog #BP220-1 into a 50 mL conical tube.
Add 35 mL PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 . Place the tube on an adjustable tilt rocker and place On ice .
Note
Larger quantities of 30% sucrose can be prepared ahead of time, filtered through a 0.22 uM filter, and stored at 4 °C for up to 1 month.
Collect about2 mm thick cross-sectional slices from the Head, Body, and Tail (see pancreas map).
Cut each slice into 4 quadrants (A, B, C, D), and drop each piece of tissue, one at a time, into the same tube.
Note
For donors less than 1 year of age, keep the entire slice intact and drop into the tube.
Transfer the tissue into a 50 mL conical tube containing 40 mL freshly prepared fixative and fix for 03:00:00 On ice under mild agitation using an adjustable tilt rocker.
3h
Wash the tissue four times in 40 mL 100 mM PBSElectron Microscopy SciencesCatalog #19344-10 over 03:00:00 . Blot the tube on a paper towel between washes.
3h
Equilibrate the tissue in 40 mL 30% sucrose solution at 4 °C Overnight . The tissue will settle to the bottom of the tube.
Note
If the pancreas is not cleaned sufficiently of fat, the tissue will not drop.
3h
Prepare a flat piece of dry ice.
Fill a pre-labeled cryomold halfway withAndwin Scientific Tissue-Tek™ CRYO-OCT CompoundFisher ScientificCatalog #14-373-65 .
Pour the pancreas with some sucrose out into a 10 cm Petri dish. Pick the tissue up with fine forceps and blot with Kimwipes to remove any excess sucrose.
Place the tissue into the prepared cryomold, and gently push toward the bottom. Add more Andwin Scientific Tissue-Tek™ CRYO-OCT CompoundFisher ScientificCatalog #14-373-65 to completely fill the mold.
Place the mold on a flat piece of dry ice. As the OCT begins to freeze, push the tissue down into the bottom of the mold again.
Once OCT is completely frozen, wrap the mold in pre-labeled piece of foil, place it in a zip-lock bag, and store at -80 °C .
Note
Aluminum foil and zip-lock bag keep the tissue from drying out.
Ship tissue blocks to Vanderbilt University Medical Center on dry ice.
Human Pancreas Processing for Clarity (CLR)
3h
Collect about2 mm thick cross-sectional slices from the Head, Body, and Tail (see figure in Step 5 ).
Cut each slice into 4 quadrants (A, B, C, D), and drop each piece of tissue, one at a time, into the same tube.
Note
For donors less than 1 year of age, keep the entire slice intact and drop into the tube.
Transfer the tissue into a 50 mL conical tube containing 40 mL freshly prepared fixative and fix for 03:00:00 On ice under mild agitation using an adjustable tilt rocker.
3h
Wash the tissue four times in 40 mL PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 over 03:00:00 . Blot the tube on a paper towel between washes.
After the last wash, add 40 mL PBS, no calcium, no magnesiumThermo Fisher ScientificCatalog #14190144 and store at 4 °C .
Ship tissue blocks to Vanderbilt University Medical Center with ice packs.
Human Pancreas Processing for Transmission Electron Microscopy (EM)
1d
Prepare TEM fixative in a 50 mL conical tube:
Note
Prepare as many tubes as the number of tissue slices to be processed.
Add 5 mL PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEEMSCatalog #15710 .
Add 10 mL 8% GlutaraldehydeElectron Microscopy SciencesCatalog #16020 .
Add 20 mL Sodium Cacodylate BufferElectron Microscopy SciencesCatalog #11652 .
Add 5 mL dH20.
Collect about2 mm thick cross-sectional slices from the Head, Body, and Tail (see figure in Step 5 ).
Place each slice into a separate 10 cm Petri dish containing 10-15 mL EM fixative at Room temperature .
Note
Cold depolymerizes cytoskeletal elements, so whenever possible fixative and sample should be at Room temperature or 37 °C to initiate fixation.
Using fine scissors, mince the sample in the plate with fixative. Alternatively, the tissue can be minced by placing it into a drop of fixative on dental wax or parafilm and using fresh razor blades.
Note
Aldehyde fixatives penetrate tissues slowly, so samples must be minced very small (less than 0.5 mm per side.
Transfer the minced tissue into a 50 mL conical tube and fix in 40 mL fixative for 24:00:00 at 4 °C under mild agitation on an adjustable tilt rocker.
1d
Ship to Vanderbilt University Medical Center within 3 days on ice packs.
The Vanderbilt group will submit the tissue in fixative to the VU EM Core for EM processing. Tissue will be embedded into Spurr resin within 7 days of fixation.
Reference Info