May 24, 2022

Public workspaceCODEX® Multiplexed Imaging | Tissue Sectioning

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyn8epvx9/v1
CODEX® Multiplexed Imaging | Tissue Sectioning
  • Diane Saunders1,
  • Alexander Hopkirk2,
  • Conrad Reihsmann2,
  • Regina Jenkins2,
  • Marcela Brissova1,
  • Alvin C. Powers1
  • 1Vanderbilt University, Vanderbilt University Medical Center;
  • 2Vanderbilt University Medical Center
  • Vanderbilt Diabetes Research Center
Islet and Pancreas Analysis Core
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyn8epvx9/v1
Protocol Citation: Diane Saunders, Alexander Hopkirk, Conrad Reihsmann, Regina Jenkins, Marcela Brissova, Alvin C. Powers 2022. CODEX® Multiplexed Imaging | Tissue Sectioning. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyn8epvx9/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: April 24, 2022
Last Modified: May 24, 2022
Protocol Integer ID: 61323
Keywords: comprehensive multiplexed imaging, tissue preparation, imaging, tissue preparation process, tissue, use at the vanderbilt diabetes research center, system by akoya bioscience, vanderbilt diabetes research center, akoya bioscience
Disclaimer
This protocol is adapted from the CODEX User Manual, revision C (Akoya Biosciences, Dec. 2020). See also: Tissue Processing - Best Practices.
Abstract
This protocol describes the tissue preparation processes for the CODEX® (now PhenoCycler™) system by Akoya Biosciences. For the comprehensive multiplexed imaging workflow currently in use at the Vanderbilt Diabetes Research Center, please see CODEX® Multiplexed Imaging | Modality overview.
Materials
ReagentPoly-l-lysine, 0.1% (wt/vol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8920
Reagent22 x 22m Glass Cover Slips # 1 1/2Electron Microscopy SciencesCatalog #72204-01


Storage:

  • ReagentPolarSafe™ PCR Cardboard Freezer BoxesFisher ScientificCatalog #10-987-065
  • Reagent 7x8 Double Zipper BagsUlineCatalog #S-20325
  • Reagent6x9 Double Zipper BagsUlineCatalog #S-17788


Equipment:
Equipment
CM1950 cryostat
NAME
Leica
BRAND
CM1950
SKU

Protocol materials
Reagent6x9 Double Zipper BagsUlineCatalog #S-17788
ReagentPoly-l-lysine, 0.1% (wt/vol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8920
Reagent22 x 22m Glass Cover Slips # 1 1/2Electron Microscopy SciencesCatalog #72204-01
ReagentPolarSafe™ PCR Cardboard Freezer BoxesFisher ScientificCatalog #10-987-065
Reagent 7x8 Double Zipper BagsUlineCatalog #S-20325
Troubleshooting
Coverslip Preparation
Gather reagents:

  • ReagentPoly-l-lysine, 0.1% (wt/vol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8920
  • Reagent22 x 22m Glass Cover Slips # 1 1/2Electron Microscopy SciencesCatalog #72204-01
Gently place coverslips at the bottom of a 500-mL glass beaker and swirl to spread the coverslips out. Add approximately Amount20 mL poly-L-lysine solution to the beaker, ensuring coverslips are fully submerged. Rotate the beaker at a 45° angle for Duration00:01:00 to mix.
Cover beaker with parafilm and incubate for minimum of Duration12:00:00 at TemperatureRoom temperature . Coverslips can sit in the poly-L-lysine solution for up to one week.
Slowly pipet off the poly-L-lysine solution and dispose.
Fill the beaker containing the coverslips to half volume with Milli-Q® water. Swirl gently, let sit for Duration00:00:30 , and then slowly pour off water into the sink.
Go to and repeat for a total of 5-7 washes.
Spread WypAll® towels on the benchtop. In small batches, remove coverslips from the beaker, separate from one another, and place individual coverslips side by side on towels. Layer another towel on top and gently dab to remove excess liquid. Leave on benchtop until completely dry.

ⓘ Coated coverslips can be stored in a petri dish for up to 2 months.
Cryosectioning
Set the cryostat temperature to Temperature-20 °C . Remove a fresh blade, clean with ethanol and a lint free wipe as needed, and insert blade.

Using OCT or CMC, mount sample onto the cryostat chuck in the desired orientation. Trim CMC using larger section increments of 30-50 μm until the tissue becomes visible (Figure 1a).


Figure 1. Sectioning cryopreserved tissue onto CODEX coverslip.
Obtain Thikness6 µm to Thikness10 µm sections, using a fine-tipped paintbrush to guide the section onto base and place coverslip onto section (Figure 1b-1d).
Using forceps, slot coverslips with mounted tissue into a plastic coverslip holder under the appropriate donor/block ID label(s).
As necessary, mount adjacent sections onto Gold Plus slides (for traditional immunohistochemistry) and/or ITO slides (for mass spectrometry). Record all sections cut, the thickness, and any additional block shaved off in the process.
Coverslip Storage
3D printed coverslip holders: Download Codex Rack MkIII-Body.stlCodex Rack MkIII-Body.stl

We use these with the following:

  • ReagentPolarSafe™ PCR Cardboard Freezer BoxesFisher ScientificCatalog #10-987-065
  • Reagent 7x8 Double Zipper BagsUlineCatalog #S-20325
  • Reagent6x9 Double Zipper BagsUlineCatalog #S-17788
Store coverslips at Temperature-80 °C for up to 6 months.