TITLE: FFPE Tissue Processing for Multimodal Imaging Assays (Phenocycler-Fusion + H&amp;E) following Xenium In Situ Gene Expression
AUTHORS: Santhosh Sivajothi, Emily Soja, Shruti Bhargava, William F Flynn, Elise T Courtois

[DESCRIPTION]
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.

[STEPS]
SECTION: Tissue Processing for Xenium In Situ Gene Expression
1. 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:

Link: CG000580 (Rev C): Xenium In Situ for FFPE Tissues – Deparaffinization & Decrosslinking
  

Link: CG000582 (Rev F): Xenium In Situ Gene Expression - Probe Hybridization, Ligation & Amplification
  

Link: CG000584 (Rev E): Xenium Analyzer UserGuide
  

SECTION: Tissue Processing for Xenium In Situ Gene Expression
1.2. The slide can be processed immediately or stored at 4 °C for up to 2 weeks.


SECTION: Slide Storage
2. For slide storage:

SECTION: Tissue Processing for Xenium In Situ Gene Expression
1.1.  At the end of the Xenium  run, remove the slide cassette assembly from the Xenium Analyzer. 

 

 


SECTION: Slide Storage
2.2. 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  

SECTION: Slide Storage
2.1. After the Xenium run, remove the liquid covering the slide. Add 1 mL PBS and incubate 5 min  Room temperature 
SECTION: Tissue processing for Phenocycler-Fusion assay
3. Tissue processing can start directly after Xenium run or from a previously stored slide
SECTION: Tissue processing for Phenocycler-Fusion assay
3.1. 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 5 min Room temperature. Repeat for a total of two washes
Wash slide in DI or Milli-Q water for 2 min Room temperature 
Proceed to step 4
SECTION: Tissue processing for Phenocycler-Fusion assay
3.2. Processing slide from storage:

Remove storage solution from the tissue and remove slide from the cassette assembly
Wash slide with PBS for 5 min Room temperature. Repeat for a total of four washes
Wash slide in DI or MilliQ water for 2 min Room temperature 
Proceed to step 4
SECTION: Tissue processing for Phenocycler-Fusion assay
4. 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.
SECTION: Tissue processing for Phenocycler-Fusion assay
4.1. 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
SECTION: Tissue processing for Phenocycler-Fusion assay
4.2. Immerse the slides in the vessel and cover with aluminum foil.
SECTION: Tissue processing for Phenocycler-Fusion assay
4.3. 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 15 min 

SECTION: Tissue processing for Phenocycler-Fusion assay
4.4. After incubation, release the pressure, remove the vessel from the cooker and rest on the bench for 30 min 
SECTION: Tissue processing for Phenocycler-Fusion assay
5. 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

  

 

SECTION: Tissue processing for Phenocycler-Fusion assay
4.5. Wash slide in DI or Milli-Q water for 2 min Room temperature 
SECTION: Tissue processing for Phenocycler-Fusion assay
6. 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.

 

 

SECTION: Flow Cell Removal
8. 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 
SECTION: Flow Cell Removal
8.1. Fill a coplin jar with xylene or Histo-Clear. There must be sufficient volume to cover the entire flow cell.
SECTION: Flow Cell Removal
8.2. Place slides in the coplin jar containing xylene or Histo-Clear and incubate 1440 min at Room temperature 
SECTION: Flow Cell Removal
8.3. Following incubation, gently remove the flow cell from the slide.

SECTION: Staining Procedure
9. Place the slides in 100% ethanol for 2 min. Raise and dip the slides 10 to 15 times to fully cover the tissue.
SECTION: Staining Procedure
10. Place the slides in 95% ethanol for 2 min. Raise and dip the slides 10 to 15 times to fully cover the tissue.
SECTION: Staining Procedure
11. Place the slides in DI water for 2 min. Raise and dip the slides 10 to 15 times to fully cover the tissue.

SECTION: Staining Procedure
13. Place the slides in the following reagent series:
SECTION: Staining Procedure
13.1. Mayer’s Hematoxylin - 4 min
 

SECTION: Staining Procedure
13.2. DI water - 1 min
SECTION: Staining Procedure
13.3. Bluing Reagent - 1 min
SECTION: Staining Procedure
13.4. DI water - 1 min
SECTION: Staining Procedure
13.5. Alcoholic Eosin – 2 min
SECTION: Staining Procedure
13.6. 95% ethanol - 1 min without agitation or 20-30 dips to remove all excess reagent
from previous step
SECTION: Staining Procedure
13.7. 100% ethanol - 1 min without agitation or 20-30 dips to remove all excess reagent from previous step
SECTION: Staining Procedure
13.8. 100% ethanol - 1 min without agitation or 20-30 dips to remove all excess reagent from previous step
SECTION: Staining Procedure
13.9. Xylene/Histo-Clear - 1 min without agitation or 20-30 dips to remove all excess reagent from previous step

SECTION: Staining Procedure
13.10. Xylene/Histo-Clear - Hold in this solution as slides are mounted one by one
SECTION: Slide Mounting
14. 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. 
SECTION: Slide Mounting
14.1. Using a disposable transfer pipette add enough DPX mountant (or other xylene-based mountants) to cover desired tissue area (1 to 2 drops) 

 

SECTION: Slide Mounting
14.2. 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. 

 

 

SECTION: Slide Mounting
14.3. Remove excess mountant by placing the edges along the paper towel. Avoid mountant covering the top of the coverslip. 

 

SECTION: Slide Mounting
14.4. Gently press down on the coverslip to remove any bubbles that remain above the stained tissue.
SECTION: Slide Mounting
15. Allow mountant to cure inside the hood for at least 20 min before moving and imaging. Xylene based mountants take up to 24 hours to cure completely.
SECTION: Slide Mounting
16.   and repeat mounting procedure for remaining slides.

SECTION: Slide Mounting
17. Mounted slides can be imaged and stored in a slide storage box at Room temperature 

 

SECTION: H&amp;E staining
7. 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.

 

SECTION: OPTIONAL: Autofluorescence Quencher Removal
12. 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
CG000613_Demonstrated_Protocol_Xenium_H_EStaining_RevB

  
SECTION: OPTIONAL: Autofluorescence Quencher Removal
12.1. Prepare 10 mL fresh quencher removal solution:
In a fume hood, weigh 17.4 mgof 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
 

SECTION: OPTIONAL: Autofluorescence Quencher Removal
12.2. IMMEDIATELY transfer the quencher removal solution to a slide mailer and place the slides into the solution. Incubate for 10 min 
SECTION: OPTIONAL: Autofluorescence Quencher Removal
12.3. Wash three times in Milli-Q water for 1 min each
SECTION: Slide Mounting
18. 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.