TITLE: 10 X Visium Spatial Gene Expression - Fixed Frozen Tissue Processing with CytAssist
AUTHORS: Alberto Pappalardo, Kavya Batra, Rolando Perez-Lorenzo, Angela Christiano

[DESCRIPTION]
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

[STEPS]
SECTION: Coverslipping
3.3. 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.
SECTION: Coverslipping
3.2. Place the coverslip without introducing bubbles and wait for the mounting medium to settle.
SECTION: Coverslipping
3. Coverslipping 
SECTION: Rehydration
1.1. Place a Low Profile Thermocycler Adapter on a thermal cycler and preheat
thermal cycler to 37°C.
SECTION: Rehydration
1.3. 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.
SECTION: Rehydration
1.4. Submerge the slide in a tube containg PBS for 5 min.
SECTION: Rehydration
1.5. Submerge the slide slowly in a new tube containing Milli-Q Water for 3 min.
SECTION: Rehydration
1.6. Submerge the slide slowly in a new tube containing 100% Ethanol for 3 min.
SECTION: Rehydration
1.7. Submerge the slide slowly in a new tube containing  70% Ethanol for 3 min.
SECTION: Rehydration
1.8. Submerge the slide slowly in a new tube containing  Milli-Q Water for 20 sec.
SECTION: Coverslipping
3.1. Remove excess water and add 100 µl mounting medium to uniformly cover the entire tissue section.
SECTION: Rehydration
1.9. Proceed immediately to H&E Staining & Coverslipping.
SECTION: Tissue Imaging
4. Imaging 

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

SECTION: Tissue Imaging
5.3. Hold the slide submerged until the coverslip slowly falls off from the slide
SECTION: Tissue Imaging
5.4. Gently dip the slide 30x in water to remove comletly the mounting medium.

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

 

SECTION: Decrosslinking
6.3. Place the slide in the Visium CytAssist Tissue Slide Cassette.
SECTION: Decrosslinking
6.4. 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.
SECTION: Decrosslinking
6.5. Remove HCl by careful aspiration.
SECTION: Decrosslinking
6.6. 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.
SECTION: Decrosslinking
6.7. Seal the cassette and place the slide on the Low Profile Thermocycler Adapter at 42°C.
SECTION: Decrosslinking
6.8. Close the thermal cycler lid and initiate Destaining.
SECTION: Decrosslinking
6.9. Remove the slide from the Low Profile Thermocycler Adapter and place on a flat surface. Some color leftover after the destaining is acceptable.
SECTION: Decrosslinking
7. Decrosslinking
SECTION: Decrosslinking
7.1. Prepare the thermal cycler with the following settings.
SECTION: Decrosslinking
7.2. Lid Temperature- 70°C
Reaction Volume - 100 µl 
Run Time - 30 min

 

SECTION: Decrosslinking
7.3. Remove all the HCl from the well corners.
SECTION: Decrosslinking
7.4. Add 150 µl of diluted decrosslinking buffer.
SECTION: Decrosslinking
7.5. Remove diluted decrosslinking buffer.
SECTION: Decrosslinking
7.6. Add 100 µl of diluted decrosslinking buffer.
SECTION: Decrosslinking
7.7. Re-seal the cassette and place the slide on the Low Profile Thermocycler Adapter at 70°C.
SECTION: Decrosslinking
7.8. Close the thermal cycler lid and initiate the decrosslinking.
SECTION: Decrosslinking
7.9. Proceed immediately to Visium CytAssist Spatial Gene Expression User Guide (CG000495).
SECTION: H&amp;E Staining
2.12. Submerge the slide 10x in a new water beaker (VI)
SECTION: H&amp;E Staining
2.11. Remove the Eosin and submerge slide for 30 sec in a new water beaker (V).
SECTION: H&amp;E Staining
2.10. 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.
SECTION: H&amp;E Staining
2.9. Remive the Bluing Buffer and submerge the slide 10x in a new water beaker (IV).
SECTION: H&amp;E Staining
2.8. 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.
SECTION: H&amp;E Staining
2.7. Submerge slide 15x in a new water beaker (III).
SECTION: H&amp;E Staining
2.6. Submerge the slide 15x in a new water beaker (II).
SECTION: H&amp;E Staining
2.5. Remove the Hematoxylin and submerge the slide 5x in a water beaker (I).
SECTION: H&amp;E Staining
2.4. Incubate for 3 min at room temperature.
SECTION: H&amp;E Staining
2.3. Cover the slide with 1 ml of Hematoxylin making sure to uniformly cover the tissue section.
SECTION: H&amp;E Staining
2.2. Place slide on a flat impermeable staining surface.
SECTION: H&amp;E Staining
2. H & E staining
SECTION: H&amp;E Staining
2.1. Prepare 6 water beaker with Milli-Q Water to dip the slide.
SECTION: Rehydration
1.2. 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.