Apr 13, 2024
FFPE Tissue Processing for Multimodal Imaging Assays (Phenocycler-Fusion + H&E) following Xenium In Situ Gene Expression
- 1Single Cell Biology Lab, The Jackson Laboratory, USA
- JAX Single Cell Biology
- Single Cell Jax
Protocol Citation: Santhosh Sivajothi, Emily Soja, Shruti Bhargava, William F Flynn, Elise T Courtois 2024. FFPE Tissue Processing for Multimodal Imaging Assays (Phenocycler-Fusion + H&E) following Xenium In Situ Gene Expression. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71rwqgwz/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 02, 2024
Last Modified: April 13, 2024
Protocol Integer ID: 97679
Keywords: Xenium, Phenocycler Fusion, multiplexed imaging, multiomics, spatial transcriptomics, spatial proteomics, codex, hematoxylin, eosin, H&E
Abstract
Xenium In Situ Gene Expression assay from 10X Genomics enables multiplexed imaging of RNA transcripts in situ. There are several antibody based multiplexed imaging technologies that enable multiplexed protein detection. Hematoxylin and Eosin (H&E) staining is a gold standard method for observing tissue morphology. In order to generate a highly multimodal imaging dataset, we developed a protocol to prepare tissue slides for multiplexed imaging on the Phenocycler-Fusion platform and H&E staining following a Xenium run.
Although the Xenium protocol includes a decrosslinking step which is akin to antigen retrieval, a second round of antigen retrieval, as outlined in this protocol, is necessary for epitope unmasking and accurate protein detection by antibody based methods.
The steps for slide storage and tissue processing for multiplexed imaging can also be applied to imaging assays on other platforms.
Materials
Specialty Reagents:
- Xenium assay kits and reagents as detailed in 10X User Guide
- Phenocycler buffers and reagents as detailed in Akoya User Guide
- Validated and barcoded antibodies for Phenocycler assay
General Reagents and Materials:
- 1X PBS, pH 7.4 (Gibco; 10010023, ThermoFisher Scientific)
- Glycerol (G5516, Sigma Aldrich)
- DI water or Milli-Q water
- Antigen retrieval buffer (pH6, Abcam ab93678 or pH9, Abcam ab93684)
- Xylene (Sigma cat# 534056)or Histo-Clear (National Diagnostics cat# HS-200)
- 100% (200 proof) ethanol - Sigma cat# E7023
- Hematoxylin Solution, Mayer′s - Sigma cat# MHS32-1L
- Epredia Shandon Bluing Reagent - Thermo cat# 6769001
- Eosin Y Solution; Alcoholic - Sigma cat# HT110116-500ML
- DPX Mountant - Sigma cat# 06522-100ML
- Coplin jars
- Slide mailers
Instrumentation:
- Xenium Analyzer (10X Genomics)
- Phenocycler-Fusion (Akoya Biosciences)
- De-cloaking chamber (Biocare Medical or BioSB TintoRetriever) or pressure cooker (Instant Pot)
- Chemical fume hood
- Weighing balance
Tissue Processing for Xenium In Situ Gene Expression
Tissue Processing for Xenium In Situ Gene Expression
FFPE tissue on Xenium slides is processed according to the protocols in 10X User Guides:
Please refer to the linked 10X User Guides for all materials and detailed instructions. The key steps are noted below:
- Tissue deparaffinization: Step 1.2 of CG000580 (Rev C)
- Decrosslinking: Step 1.4 CG000580 (Rev C). We selected the 80 °C condition that is recommended for all tissues other than skin.
- Probe hybridization: step 1 of CG000582 (Rev F)
- Post hybridization wash: step 2 of CG000582 (Rev F)
- Ligation: step 3 of CG000582 (Rev F)
- Amplification: step 4 CG000582 (Rev F)
- Autofluorescence quenching and nuclear staining: step 5 of CG000582 (Rev F)
After processing, the slide was loaded onto the Xenium Analyzer instrument and run according to instructions in User Guide CG000584 (Rev E)
User Guides:
CG000580_Demonstrated_Protocol_Xenium_FFPE_Deparaffinization_Decrosslinking_RevC.pdf7MB 10x_CG000582_XeniumInSitu_GeneExpression_UserGuide_RevF.pdf7.9MB CG000584_Xenium_Analyzer_UserGuide_RevE.pdf13MB At the end of the Xenium run, remove the slide cassette assembly from the Xenium Analyzer.
Xenium slide cassette assembly
Note
The cassette assembly can be retained for use in future incubation steps
The slide can be processed immediately or stored at 4 °C for up to 2 weeks.
Slide Storage
Slide Storage
For slide storage:
After the Xenium run, remove the liquid covering the slide. Add 1 mL PBS and incubate 00:05:00 mins Room temperature
5m
Remove PBS. Add 1 mL storage solution (50% glycerol in PBS) to the slide. Replace the lid to seal the cassette and store for up to 2 weeks at 4 °C
Tissue processing for Phenocycler-Fusion assay
Tissue processing for Phenocycler-Fusion assay
1h 1m
1h 1m
Tissue processing can start directly after Xenium run or from a previously stored slide
Processing slide directly after Xenium run:
- After the Xenium run, remove the liquid from the slide and remove slide from the cassette assembly
- Wash slide in PBS for 00:05:00 mins Room temperature . Repeat for a total of two washes
- Wash slide in DI or Milli-Q water for 00:02:00 mins Room temperature
- Proceed to step 4
7m
Processing slide from storage:
- Remove storage solution from the tissue and remove slide from the cassette assembly
- Wash slide with PBS for 00:05:00 mins Room temperature . Repeat for a total of four washes
- Wash slide in DI or MilliQ water for 00:02:00 mins Room temperature
- Proceed to step 4
7m
Perform antigen retrieval to de-crosslink and reveal epitopes. Antigen retrieval can be performed in citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0). Antigen retrieval buffers are readily available from several vendors. Choose the retrieval buffer that is compatible with the entire panel of antibodies used in the multiplexed imaging assay.
Prepare the requisite amount of 1X antigen retrieval buffer by diluting in Milli-Q water. Ensure the vessel holding the slides contains enough buffer to cover the sample slide
Immerse the slides in the vessel and cover with aluminum foil.
Fill the pressure cooker with approximately 500 mL DI water and place the vessel containing the slides inside. Close the pressure cooker and set to the low-pressure setting ( 90 °C -110 °C ) for 00:15:00 mins
15m
After incubation, release the pressure, remove the vessel from the cooker and rest on the bench for 00:30:00 mins
30m
Wash slide in DI or Milli-Q water for 00:02:00 mins Room temperature
2m
After this point, continue processing by following the protocol from Chapter 3, page 50, Step 4 in the PhenoCycler-Fusion User Guide PD-000011 Rev L
PhenoCycler-Fusion User Guide_2.1.0.pdf15.3MB Note
To continue immunostaining via other assays, transfer slide to PBS and follow the corresponding protocol steps.
Expected result:
Human pancreas tissue was processed for Xenium in situ gene expression assay using a custom probe set. Following Xenium run, the tissue was processed for multiplexed imaging on Phenocycler-Fusion using a panel of 40 antibodies following the steps listed in this protocol.
Image of human pancreas tissue visualized in Xenium Explorer showing RNA transcript expression overlaid on top of protein expression data. (Data courtesy of Prof. Paul Robson, The Jackson Laboratory)
Animation demonstrating transcript expression (Xenium) and protein expression (Phenocycler-Fusion) data in normal human pancreas tissue (Data courtesy of Prof. Paul Robson, The Jackson Laboratory)
H&E staining
H&E staining
After a successful Phenocycler run, slides with attached flow cells can be stored in the Akoya Storage Buffer at 4 °C or used immediately for H&E staining.
Note
Tissues that have been processed for Xenium assay have undergone autofluoresence quenching. 10X user guides recommend quencher removal before proceeding to H&E staining. In our tests, we found that autofluoresence quencher removal is not critical for good quality H&E staining.
If desired, autofluoresence quencher removal can be performed (Step 12)
Flow Cell Removal
Flow Cell Removal
1h 1m
1h 1m
The flow cell needs to be removed from the slide to continue with H&E staining. Retrieve tissue slides with attached flow cells either from the storage buffer or directly after Phenocycler run
Fill a coplin jar with xylene or Histo-Clear. There must be sufficient volume to cover the entire flow cell.
Place slides in the coplin jar containing xylene or Histo-Clear and incubate 24:00:00 hours at Room temperature
1d
Following incubation, gently remove the flow cell from the slide.
Staining Procedure
Staining Procedure
1h 1m
1h 1m
Place the slides in 100% ethanol for 00:02:00 mins . Raise and dip the slides 10 to 15 times to fully cover the tissue.
2m
Place the slides in 95% ethanol for 00:02:00 mins . Raise and dip the slides 10 to 15 times to fully cover the tissue.
2m
Place the slides in DI water for 00:02:00 mins . Raise and dip the slides 10 to 15 times to fully cover the tissue.
2m
OPTIONAL: Autofluorescence Quencher Removal
OPTIONAL: Autofluorescence Quencher Removal
If autofluoresence quencher removal is desired, it can be performed at this stage before proceeding to Step 13 for staining.
For full details, check 10X Genomics User Guide CG000613
10x_CG000613_Demonstrated_Protocol_Xenium_H_EStaining_RevB.pdf2MB Prepare 10 mL fresh quencher removal solution:
- In a fume hood, weigh 17.4 mg of sodium hydrosulfite
- Add the sodium hydrosulfite into 10 mL of Milli-Q water in a 15 mL conical tube. Close the tube and vortex briefly until dissolved
Note
The quencher removal solution is only effective for 00:10:00 mins and must be used immediately. If the solution turns white, prepare fresh.
IMMEDIATELY transfer the quencher removal solution to a slide mailer and place the slides into the solution. Incubate for 00:10:00 mins
10m
Wash three times in Milli-Q water for 00:01:00 min each
1m
Staining Procedure
Staining Procedure
1h 1m
1h 1m
Place the slides in the following reagent series:
Mayer’s Hematoxylin - 00:04:00 mins
Note
Lymphoid tissues or tissues with dense cellularity should be stained for only 00:03:00 mins . Rinse the slide in DI water to observe the stain. Return the slide to Mayer's hematoxylin if staining is not adequate.
4m
DI water - 00:01:00 min
1m
Bluing Reagent - 00:01:00 min
1m
DI water - 00:01:00 min
1m
Alcoholic Eosin – 00:02:00 mins
2m
95% ethanol - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent
from previous step
1m
100% ethanol - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent from previous step
1m
100% ethanol - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent from previous step
1m
Xylene/Histo-Clear - 00:01:00 min without agitation or 20-30 dips to remove all excess reagent from previous step
1m
Xylene/Histo-Clear - Hold in this solution as slides are mounted one by one
Slide Mounting
Slide Mounting
20m
20m
To mount the slides, remove slides one at a time on to a paper towel inside the fume hood. Leave other slides in the last xylene/Histo-Clear coplin jar to prevent over-drying the tissue.
Dry the back of the slide and tilt to remove excess xylene, but do not let the tissues dry.
Using a disposable transfer pipette add enough DPX mountant (or other xylene-based mountants) to cover desired tissue area (1 to 2 drops)
Clean a glass coverslip quickly and remove any particles on its surface. Place the long edge of the coverslip on the edge of the slide closest to you, tip the slide towards yourself so the mountant begins to reach the coverslip, and gently push down until the mounting media has spread evenly across the slide area containing tissue.
Remove excess mountant by placing the edges along the paper towel. Avoid mountant covering the top of the coverslip.
Gently press down on the coverslip to remove any bubbles that remain above the stained tissue.
Allow mountant to cure inside the hood for at least 00:20:00 mins before moving and imaging. Xylene based mountants take up to 24 hours to cure completely.
20m
go to step #14 and repeat mounting procedure for remaining slides.
Mounted slides can be imaged and stored in a slide storage box at Room temperature
H&E staining of human FFPE pancreas tissue following Xenium and Phenocycler-Fusion assays (Data courtesy of Prof. Paul Robson, The Jackson Laboratory)
Expected Result:
Animation showing the expected result following each assay- Xenium, followed by Phenocycler-Fusion, followed by H&E staining on the same FFPE pancreas tissue.
(Data courtesy of Prof. Paul Robson, The Jackson Laboratory)