Mar 19, 2024

Public workspace10 X Visium Spatial Gene Expression - Fixed Frozen Tissue Processing with CytAssist

DOI
dx.doi.org/10.17504/protocols.io.q26g71z49gwz/v1
  • 1Columbia University
kavya Batra
Open access
DOI: dx.doi.org/10.17504/protocols.io.q26g71z49gwz/v1
External link: https://cdn.10xgenomics.com/image/upload/v1680564483/support-documents/CG000662_Demonstrated_Protocol_VisiumCytAssist_FixedFrozen_H_E_RevA.pdf
Protocol Citation: Alberto Pappalardo, Kavya Batra, Rolando Perez-Lorenzo, Angela Christiano 2024. 10 X Visium Spatial Gene Expression - Fixed Frozen Tissue Processing with CytAssist. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71z49gwz/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: March 18, 2024
Last Modified: March 19, 2024
Protocol Integer ID: 96879
Funders Acknowledgement:
NCI
Grant ID: 5UG3CA275686
Abstract
Here we summarize the reccomendation released by 10X Genomics for processing fixed-frozen tissue sections while performing 10X Visium Spatial Gene Expression assay using the Visium CytAssist device. Please follow the link for the detailed official protocol on 10X Genomics website. https://cdn.10xgenomics.com/image/upload/v1680564483/support-documents/CG000662_Demonstrated_Protocol_VisiumCytAssist_FixedFrozen_H_E_RevA.pdf
Materials
Reagents
Visium FFPE Reagent Kit PN-1000436 Store at -20°C
AB
Item Part number
Amp Mix B2000567
Extension Enzyme2000389
Extension Buffer2000409
RNase Enzyme3000593
RNase Buffer B2000551
Tissue Removal Enzyme3000387
Tissue Removal Buffer B2000543
Tissue Removal Buffer Enhancer2000557
Decrosslinking Buffer2000566
TS Primer Mix B2000537
Block and Stain Buffer 2000554
Visium FFPE Reagent Kit PN-1000436

Other Specific Reagents
ABCD
ItemAlternatives/Options Vendor Part Number
EthanolEthyl Alcohol, 200 ProofMillipore Sigma E7023
Ethanol absolute ≥99.5%VWR83813.360DP
Eosin Eosin Y-solution, AlcoholicMillipore Sigma HT110116
HematoxylinHematoxylin Solution, Mayer'sMillipore SigmaMHS16
Bluing ReagentBluing Reagent, DakoAgilentCS70230-2
1X PBS Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4Corning21-040-CV
GlycerolGlycerol SolutionMillipore Sigma49781
0.1 N HClHydrochloric Acid Solution, 0.1 NFisher Chemical SA54-1

Other Materials
Visium CytAssist Slide and Cassettes, 6.5 mm PN-1000519 for 2 runs Store at ambient temperature
ABC
Item Quantity Part number
Visium Cassette, 8 port13000811
Visium Tissue Slide Cassette
Visium CytAssist moveable gasket small (pre-assembled with translator)2 3000814
Visium CytAssist moveable translator (pre-assembled with gasket)23000816
Visium CytAssist moveable Cassette, frame23000813
Visium CytAssist Slide Seals, 40 pack1 2000284
Visium CytAssist Spatial Gene Expression Slide v2, 6.5 mm 12000549
Visium CytAssist Slide and Cassettes, 6.5 mm PN-1000519

Equipment Thermal Cycler
Low Profile Plate Insert PN-3000823 10x Magnetic Separator PN-120250


Rehydration
Rehydration
Rehydration
Place a Low Profile Thermocycler Adapter on a thermal cycler and preheat thermal cycler to 37°C.
Retrieve the slides holding the tissue from the -80 ºC freezer. The tissue sections should be 10 µm thick and placed within the allowable area for the CytAssist device. For Fisherbrand Superforst slides the allowable area is a rectangle with the lenght sides distant 5 mm from the slide edge, and with the width sides distant 15 mm from the frosted area and the plus signs.
Place slide on the Low Profile Thermocycler Adapter with the tissue side facing up. Dry for 10 min at 37°C keeping the thermal cycler lid open.
Submerge the slide in a tube containg PBS for 5 min.
Submerge the slide slowly in a new tube containing Milli-Q Water for 3 min.
Submerge the slide slowly in a new tube containing 100% Ethanol for 3 min.
Submerge the slide slowly in a new tube containing 70% Ethanol for 3 min.
Submerge the slide slowly in a new tube containing Milli-Q Water for 20 sec.
Proceed immediately to H&E Staining & Coverslipping.
H&E Staining
H&E Staining
H & E staining
Prepare 6 water beaker with Milli-Q Water to dip the slide.
Place slide on a flat impermeable staining surface.
Cover the slide with 1 ml of Hematoxylin making sure to uniformly cover the tissue section.
Incubate for 3 min at room temperature.
Remove the Hematoxylin and submerge the slide 5x in a water beaker (I).
Submerge the slide 15x in a new water beaker (II).
Submerge slide 15x in a new water beaker (III).
Place slide on back on the staining surface and add 1 ml of Bluing Buffer making sure to uniformly cover the tissue section. Incubate 1 min at room temperature.
Remive the Bluing Buffer and submerge the slide 10x in a new water beaker (IV).
Place slide on back on the staining surface and add 1 ml of Eosin making sure to uniformly cover the tissue section. Incubate 1 min at room temperature. DO NOT use diluted Eosin.
Remove the Eosin and submerge slide for 30 sec in a new water beaker (V).
Submerge the slide 10x in a new water beaker (VI)
Coverslipping
Coverslipping
Coverslipping
Remove excess water and add 100 µl mounting medium to uniformly cover the entire tissue section.
Place the coverslip without introducing bubbles and wait for the mounting medium to settle.
Once the coverslip settles, immediately proceed with imaging or store slide laying flat at 4°C in the dark for up to two weeks. If storing multiple slides avoid any contact between slides. DO NOT exceed two weeks of storage time.
Tissue Imaging
Tissue Imaging
Imaging

Image each tissue section individually at the desired magnification using brightfield imaging settings (400x reccomended).
After imaging, proceed immediately to Coverslip Removal or store as described above. DO NOT exceed two weeks of storage time.
Coverslip Removal
Fill a beaker with 800 ml of Milli-Q water (replace the water after processing 10 slides).
Submerge the slide holding it parallel to the surface and with the coverslipped surface facing sideways.

Hold the slide submerged until the coverslip slowly falls off from the slide
Gently dip the slide 30x in water to remove comletly the mounting medium.

Let the slide air dry for a minimum of 5 min until the tissue is mostly dry. DO NOT exceed 20 min.
Incubate slide on the Low Profile Thermocycler Adapter with the thermal cycler lid open for 3 min at 37°C to dry the slide.
Proceed immediately to Decrosslinking.
Decrosslinking
Decrosslinking
Destaining
Place a Low Profile Thermocycler Adapter in the thermal cycler and set the following parameters.
Lid Temperature- 42°C
Reaction Volume - 100 µl 15
Run Time - 15 min

ABC
StepTemperatureTime
Pre-equilibrate42°C Hold
Destaining42°C00:15:00
Hold22°CHold
Incubation protocol for thermal cycler

Place the slide in the Visium CytAssist Tissue Slide Cassette.
Add 150 µl 0.1 N HCl in a 6.5mm gasket along the side of the wells to uniformly cover the tissue sections (DO NOT introduce bubbles) and ensure uniform coverage.
Remove HCl by careful aspiration.
Add 100 µl 0.1 N HCl along the side of the wells to uniformly cover the tissue sections (DO NOT introduce bubbles) and ensure uniform coverage.
Seal the cassette and place the slide on the Low Profile Thermocycler Adapter at 42°C.
Close the thermal cycler lid and initiate Destaining.
Remove the slide from the Low Profile Thermocycler Adapter and place on a flat surface. Some color leftover after the destaining is acceptable.
Decrosslinking
Prepare the thermal cycler with the following settings.
Lid Temperature- 70°C
Reaction Volume - 100 µl
Run Time - 30 min

ABC
Step TemperatureTime
Pre-equilibrate70°C Hold
Decrosslinking70°C00:30:00
Cooling22°C00:10:00
Hold22°CHold
Incubation protocol for thermal cycler

Remove all the HCl from the well corners.
Add 150 µl of diluted decrosslinking buffer.
Remove diluted decrosslinking buffer.
Add 100 µl of diluted decrosslinking buffer.
Re-seal the cassette and place the slide on the Low Profile Thermocycler Adapter at 70°C.
Close the thermal cycler lid and initiate the decrosslinking.
Proceed immediately to Visium CytAssist Spatial Gene Expression User Guide (CG000495).