TITLE: HuBMAP | VU TMC Eye/Pancreas | Human Pancreas Processing for Multiple Applications
AUTHORS: Diane  Saunders, Rita  Bottino, Marcela  Brissova, Angela  Kruse, Jamie  Allen, Carrie   Romer, Danielle  Gutierrez, Jeff  Spraggins, Alvin C. Powers

[DESCRIPTION]
This protocol describes the workflow for collecting human pancreas samples for multimodal downstream assays, in use by the Vanderbilt University HuBMAP Tissue Mapping Center.

[STEPS]
SECTION: Organ Dissection and Cross-Sectioning
4. Dissect the pancreas by carefully removing duodenum, mesentery, fat, and spleen (Figure 1).


   

⚠ Note: If time and personnel permit and there are pieces of duodenum and/or spleen, retain and freeze as tissue blocks (see Flash Freezing subheading below).
SECTION: Organ Dissection and Cross-Sectioning
5. Once the pancreas is cleaned, blot with a sterile gauze square and quickly transfer to a clean plastic weigh boat. Place on a pre-tared balance and record pancreas weight.
SECTION: Organ Dissection and Cross-Sectioning
6. Immediately transfer the pancreas on an ice-cooled dissecting platform and take a photograph with a ruler as shown in Figure 2. Record pancreas size.

  
SECTION: Organ Dissection and Cross-Sectioning
7. Apply   to length of organ as follows:

Anterior: blue
Superior: black
Posterior: green
Inferior: red


   
SECTION: Formalin Fixation and Paraffin Embedding (PE)
12. Place all tissue quadrants from one disc into a 50-mL Falcon tube containing 40 mL of  .
SECTION: Formalin Fixation and Paraffin Embedding (PE)
14. Wash tissue four times in 40 mL of 100 mM PBS at 4 °C for a period of 180 min, rocking. Blot the tube with paper towel before adding the fresh washing solution.
SECTION: Formalin Fixation and Paraffin Embedding (PE)
15. After the last wash, add 40 mL of 70% ethanol and store at 4 °C.
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
17. Place all tissue quadrants from one disc into a 50-mL Falcon tube containing 40 mL of freshly prepared fixative (4% PFA*/100 mM PBS).

* is used; see step #2 for preparation details.
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
19. Wash tissue four times in 40 mL of 100 mM PBS on ice for a period of 2-3 hours (30 min per wash), rocking. Blot the tube with paper towel before adding the fresh washing solution.
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
20. After the last wash, equilibrate the tissue in 40 mL of sucrose solution (30% sucrose/10 mM PBS) at 4 °C 45 min. Tissue will settle to bottom of the tube.
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
21. Fill a prelabeled with a thin layer of carboxymethylcellulose (CMC). Pour tissue samples and sucrose solution from 50-mL tube into a 10-cm petri dish. Using the photographs described in step #10 and tissue marking dyes, identify quadrants A-D.
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
23. Transfer the cryomold to a dry ice block to freeze, adding more CMC to completely fill the indentation. When CMC compound is completely frozen, wrap each tissue block tightly in a pre-labeled aluminum foil and place it a storage bag to prevent it from drying out.
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
24. Store samples at -80 °C.

▷ If sample transfer is required, ship blocks on dry ice.


SECTION: Flash Freezing +/- CMC Embedding (FF, FCMC)
26. Place each tissue quadrant into a prelabeled   and transfer to isopentane slurry. Cover box with lid and allow tissue to completely freeze.
SECTION: Flash Freezing +/- CMC Embedding (FF, FCMC)
27. Place all wrapped samples in prelabeled storage bags and store at -80 °C.

▷ If sample transfer is required, ship blocks on dry ice.


SECTION: Preservation for Spatial Transcriptomics (RNA)
28. Place all tissue quadrants from one disc into a 50-mL Falcon tube with approximately 5 volumes of
  (four quadrants ≈ 3.5 g or 18 mL).
SECTION: Preservation for Spatial Transcriptomics (RNA)
29. Store samples at 4 °C.

▷ If sample transfer is required, ship sealed tubes on ice packs.


1. Prepare reagents, tubes, labels, etc. as outlined in the template linked below.
 

2. Prepare solutions as follows.

100 mM PBS, 1L: Add 12.07 g Na2HPO4 (dibasic), 2.04 g KH2PO4 (monobasic), 8.0 g NaCl, and 2.0 g KCl to 1L glass bottle, then add MilliQ water to volume. Place on stir plate until dissolved. Filter through a 0.22 μm filter and store at 4ºC for up to 1 month.

70% Ethanol, 1L: Combine 700 mL of 190 Proof ethanol with 300 mL of dH2O.

16% Paraformaldehyde (PFA), for FCMC: Add 60 mL of 16% PFA stock into a 500 mL glass bottle, then add 180 mL of 100 mM PBS. Mix gently and transfer 40-mL aliquots into prelabeled 50-mL conical tubes for fixation.
       ⚠ This solution should be made fresh within 12 hours of use.

16% Paraformaldehyde (PFA), for CLR: Add 20 mL of freshly-opened 32% PFA ampule into a 500 mL glass bottle, then add 140 mL of 100 mM PBS. Mix gently and transfer 30-mL aliquots into prelabeled 50-mL conical tubes for fixation.
       ⚠ This solution must be made fresh immediately prior to use.

30% Sucrose: Add 72 g sucrose to approximately 100 mL of 10 mM PBS in a 500 mL glass bottle. Place on stir plate until dissolved, then bring to 240 mL total volume with 10 mM PBS. Filter through a 0.22 μm filter and store at 4ºC for up to 1 month.

2.6% Carboxymethylcellulose (CMC): Add 500 mL water to 500 mL glass bottle and microwave for 2 minutes. Place bottle on heated stir plate (~70ºC), and slowly add 13g CMC to warmed water. Stir on low hotplate overnight, occasionally tightening lid and shaking bottle. Store at 4ºC for up to 2 months. Before each use, pour solution into 125 mL bottle and sonicate for 10 minutes.

10% Neutral Buffered Formalin: Pipet 18-mL aliquots into prelabeled 50-mL conical tubes.

RNAlater™ Stabilization Solution: Pipet 40-mL aliquots into prelabeled 50-mL conical tubes.

SECTION: Formalin Fixation and Paraffin Embedding (PE)
16. To process for paraffin embedding, transfer fixed tissue samples from 50-mL tube into a 10-cm petri dish. Using the photographs described in step #10 and tissue marking dyes, identify quadrants A-D. Place each quadrant, medial side down, into prelabeled  .
SECTION: Organ Dissection and Cross-Sectioning
8. Once the pancreas has been marked, start collecting approximately   cross-sectional slices ("discs") starting from the head (duodenum) and moving laterally towards the tail (spleen) as outlined in Figure 4. Each disc should contain dye markings on the exterior to provide spatial orientation.

   
SECTION: Organ Dissection and Cross-Sectioning
9. Photograph each disc, medial side down, using prelabeled cards with nomenclature developed for Vanderbilt LIMS (encodes positional information and processing type).

As discs are photoraphed, make notes on the experimental spreadsheet if any of the following gross morphology is observed:

Dilated ducts, cystic change, or stones
Hemorrhage
Tissue breakdown (necrosis)
Chalk-white areas (calcification)
Visible neoplasms
Other features
SECTION: Organ Dissection and Cross-Sectioning
10. Divide each disc into four approximately equal-sized quadrants, attempting to bisect dye markings so that each tissue piece contains 2 dye colors (Figure 5). Photograph disc again after quadrants are cut.

  
SECTION: Organ Dissection and Cross-Sectioning
11. Transfer each set of quadrants to the appropriate processing protocol as outlined in Figure 4.
SECTION: PFA Fixation for Tissue Clearing (CLR)
30. Place all tissue quadrants from one disc into a 50-mL Falcon tube containing 40 mL of freshly prepared fixative (4% PFA*/0.1 M PBS). 

* stock is used; see step #2 for preparation details.
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
22. Pick each quadrant up with a pair of fine forceps, blot on a Kimwipe to remove excess sucrose, and place into the appropriate CMC-prepared cryomold. Using the forceps, push the tissue lightly to bottom of the cryomold, medial side down.
SECTION: Organ Dissection and Cross-Sectioning
3. Upon arrival, remove the pancreas from transport solution (UW or HTK) and transfer into a large dissection tray placed on ice. Photograph the organ and note the following prior to dissection:

Gross morphology:

Peri-pancreatic fat: normal or excessive?
Peri-pancreatic adhesions: yes or no?
Texture: soft, firm, or hard? Any palpable masses?
Outer surface: is contour smooth or irregular? Note any localized bulge, hemorrhage, breakdown, chalk-white areas, or changes in color.
Other external features: describe as needed.
SECTION: Flash Freezing +/- CMC Embedding (FF, FCMC)
25. Prepare isopentane slurry:

Place a layer of dry ice into an insulated, lidded container and add enough 
  (isopentane) to make a slurry.
SECTION: Formalin Fixation and Paraffin Embedding (PE)
13. Fix   or at least 1080 min at 4 °C under mild agitation using an adjustable tilt rocker (LabNet).
SECTION: Light PFA Fixation and CMC Embedding (PCMC)
18. Fix for 180 min on ice under mild agitation using an adjustable tilt rocker (LabNet).

SECTION: PFA Fixation for Tissue Clearing (CLR)
31. Fix for 960 min at 4 °C under mild agitation using an adjustable tilt rocker (LabNet).

SECTION: PFA Fixation for Tissue Clearing (CLR)
33. Continue immediately to tissue clearing protocol, or store samples at 4 °C in 100 mM PBS containing 0.1% sodium azide.
SECTION: PFA Fixation for Tissue Clearing (CLR)
32. Wash tissue three times in 40 mL of 100 mM PBS on ice under mild agitation.
SECTION: Flash Freezing +/- CMC Embedding (FF, FCMC)
26.1. For FF samples (no embedding), wrap frozen tissue samples tightly in prelabeled squares of aluminum foil.
SECTION: Flash Freezing +/- CMC Embedding (FF, FCMC)
26.2. For FCMC, apply a thin layer of CMC into prelabeled  on the isopentane slurry. Allow the CMC to begin freezing (noted by turning opaque) around the edges before placing tissue quadrant into the mold. 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 prelabeled square of aluminum foil.
SECTION: Formalin Fixation and Paraffin Embedding (PE)
16.1. Follow schedule as follows using Epredia™ Excelsior™ AS Tissue Processor (total 16 hours):

70% Ethanol, one change - 45 min 
90% Ethanol, one change - 45 min
95% Ethanol, one change - 45 min
100% Ethanol, three changes - 35 min each
Xylene or xylene substitute (e.g., Clear Rite 3), three changes - 35 min each
Paraffin wax, 58 °C to 60 °C, three changes - 60 min each
SECTION: Formalin Fixation and Paraffin Embedding (PE)
16.2. Trim blocks as necessary and store at Room temperature.