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

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

Prepare 6 water beaker with Milli-Q Water to dip the slide.

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)

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).

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.

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.

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

A	B	C
Step	Temperature	Time
Pre-equilibrate	42°C	Hold
Destaining	42°C	00:15:00
Hold	22°C	Hold

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

A	B	C
Step	Temperature	Time
Pre-equilibrate	70°C	Hold
Decrosslinking	70°C	00:30:00
Cooling	22°C	00:10:00
Hold	22°C	Hold

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).