Question: Are fixed tissues embedded in OCT compatible with Xenium?
Answer: Below is a summary of our learnings and considerations from working with fixed frozen (FxF) tissue, mouse brain and kidney. We cannot guarantee assay performance even when implementing the guidance listed in this article and therefore, customers should proceed at their own risk. 10x Genomics Support does not have additional information beyond what is provided in this article and will not be able to provide additional details from internal experiments or replacements for poor assay performance.
Xenium In Situ Gene Expression can theoretically analyze mRNA in tissue sections derived from fixed frozen tissue samples, but officially unsupported. Proper tissue handling and preparation techniques preserve the morphological quality of the tissue sections and the integrity of mRNA transcripts is critical for the downstream assay workflow.
Here we provide an overview of:
- Best practices for block preparation (CG000663)
- Sectioning (CG000579)
- Sample preparation
Xenium FxF Block Preparation and Sectioning
The methods presented here are adapted from the Visium CytAssist Spatial Gene Expression for Fixed Frozen – Tissue Preparation Guide (CG000663).
- Harvest fresh tissue & quickly rinse with cold 1x PBS to remove any residual blood.
- If using mouse tissue, perfuse with 4% PFA and proceed immediately with tissue fixation.
- Tissue Fixation
- Drop-fix in cold 4% PFA overnight (12-16 hrs). Ensure the sample is fully submerged in fixative and has sunk to the bottom.
- Before proceeding with cryopreservation, wash with 1x PBS 3 times
- Two approaches: sucrose gradient (15%-30% sucrose) or dropping sample into 30% sucrose solution for approximately 6-12 hrs (or until tissue sinks).
- Embed in OCT
- Utilize isopentane bath and rapid freeze sample in OCT-filled mold.
The methods presented here are adapted from the Xenium In Situ for Fresh Frozen Tissues - Tissue Preparation Guide (CG000579).
‘!’ Tip: Recommend identifying optimal cutting temperature for sample prior to mounting on Xenium slides. Fixation may impact ease of sectioning and may require cooler than normal temperatures (compared to fresh frozen tissues).
‘!’ Tip: Equilibrate Xenium slides prior to collection by storing in CryoChamber 15-30min before sectioning.
- As the sample is already in OCT, skip Chapter 1 on Tissue Embedding and proceed to block QC.
- We recommend block QC prior to sectioning on Xenium slides by H&E (Chapter 2).
- After blocks are QC’d, proceed to sectioning according to the Xenium In Situ for Fresh Frozen Tissues - Tissue Preparation Guide (CG000579).
- Once slides are sectioned, the next step is sample preparation.
- It is possible to store sectioned slides at -80°C (two slides in a slide mailer) for up to four weeks.
- Rehydration (CG000662). There are minor modifications to the protocol listed below.
- Decrosslinking (CG000580)
Detailed Sample Preparation Protocol
1.1 Rehydration Buffer Preparation (CG000662)
Prepare all buffers fresh according to the tables below before retrieving tissue sections from -80°C.
Prepare buffers in appropriate sized conical tube or bottle and transfer carefully to corresponding slide mailer.
a. Prepare 1X PBS. Add reagents in the order listed. Invert gently to mix. Maintain at room temperature. This volume of 1X PBS is sufficient for washes in all subsequent steps of this Demonstrated Protocol.
b. Using 1X PBS, prepare PBS-Tween (PBS-T). Add reagents in the order listed. Invert gently to mix. Maintain at room temperature. This volume of PBS-T is sufficient for washes in subsequent steps of this Demonstrated Protocol.
c. Start thawing Xenium FFPE Tissue enhancer (used for decrosslinking, step 1.3b). Thaw in a thermomixer for 30 mins at 37°C, 300 rpm with shaking. Cool to room temperature for 5 minutes. Vortex for 30 sec and centrifuge briefly.
1.2 Thawing and rehydration (CG000662)
a. Place Xenium Thermocycler Adaptor in thermal cycler set to incubate at 37°C. DO NOT close the lid. Prepare the 1X PBS Mailer and a timer set to 1 min, which are needed in the following steps.
b. Prepare an ice bucket of dry ice.
c. Remove slide mailer containing stored fixed frozen tissue slide(s) from -80°C and bury into the dry ice.
d. Using a pair of slide forceps, move the slide(s) from dry ice to the 37°C pre-heated thermal cycler for 1 min.
‘!” This step is modified from the Visium FxF sample preparation protocol.
e. Place the slide on the Xenium Thermocycler Adaptor with the tissue side facing up. Ensure the Sample Area is aligned with the corresponding area on the Thermocycler Adaptor. DO NOT close the lid.
f. Immediately remove slide from thermal cycler following incubation. Gently immerse the slide in the 1X PBS Mailer using slide forceps and incubate for 5 min at room temperature.
g. Gently immerse slide in the Milli-Q Water Mailer 1 and incubate for 3 min at room temperature.
h. Gently immerse slide in the 100% Ethanol Mailer for 3 min at room temperature.
i. Gently immerse slide in the 70% Ethanol Mailer for 3 min at room temperature.
j. Gently immerse slide in the Milli-Q Water Mailer 2 for 20 sec at room temperature.
k. Remove slide from the Milli-Q Water Mailer 2.
l. Remove any remaining Milli-Q Water from the slide using a lint-free laboratory wipe. Dry back of slide completely and front of slide outside of Sample Area without touching or damaging the tissue sections. Place The Slide In the Xenium Cassette. Refer to Tips Best Practices for guidance Xenium Cassette Assembly.
‘!’ Work quickly to avoid tissue sections drying.
m. Add 500μl PBS-T.
‘!” Do not proceed to H&E coverslipping.
1.3 Decrosslinking Buffer Preparation
Prepare all buffers fresh according to the tables below.
a. Prepare Diluted Perm Enzyme B using 1X PBS prepared from step 1.1a. Add reagents in the order listed. Mix thoroughly with a 1-ml pipette set to 600μl. Maintain at room temperature.
b. Prepare Decrosslinking Buffer using Xenium FFPE Tissue Enhancer prepared at 1.1c and Diluted Perm Enzyme B prepared at step 1.3a. Add reagents in the order listed. Pipette mix thoroughly. Maintain at room temperature in the dark.
1.4 Decrosslinking (CG000580)
Reagent addition and removal should be done carefully. Remove reagents along the side of the well without touching the tissue sections and without introducing bubbles.
a. Place a Xenium Thermocycler Adaptor in the thermal cycler.
b. Prepare the thermal cycler with the following incubation protocol and start the program.
c. Remove 1X PBS-T from Xenium Cassette well.
d. Add 500 μl Decrosslinking Buffer along the side of the well to uniformly cover the tissue sections, without introducing bubbles. Tap Xenium Cassette gently to ensure uniform coverage.
e. Apply a new Xenium Cassette Lid on the Xenium Cassette and place the cassette on the Thermocycler Adaptor at 22°C. Close the thermal cycler lid.
f. Skip Hold step and initiate Decrosslinking.
'!' Start thawing reagents for Probe Hybridization during Decrosslinking incubation as indicated in Xenium Situ Gene Expression-Probe Hybridization, Ligation & Amplification User Guide (CG000582).
g. Remove Xenium Cassette from the thermal cycler and place on a flat, clean work surface.
h. Remove the Xenium Cassette Lid and using a pipette, remove all Decrosslinking Buffer from the well. Discard old Cassette Lids.
i. Add 500 μl PBS-T to the well.
j. Incubate for 1 min at room temperature.
k. Carefully remove all PBS-T from the well using a pipette tip.
l. Repeat steps i-k two more times.
m. Add 500 μl PBS-T to the well.
n. Proceed immediately to Xenium In Situ Gene Expression - Probe Hybridization, Ligation & Amplification User Guide (CG000582).
Xenium Assay Workflow
No modifications are made to the Xenium In Situ Gene Expression - Probe Hybridization, Ligation & Amplification workflow (CG000582) for FxF samples.
Product: Xenium In Situ Gene Expression