H&E Staining for 10X Genomics Visium Imaging

Prechill methanol (40mL/slide, dispensed in a 50-mL centrifuge tube) to -20°C .

Place a Thermocycler Adaptor on a thermal cycler set at 37°C and equilibrate for 0h 5m 0s . Heating the Thermocycler lid is not required.

Remove slide from -80°C and place on dry ice in a sealed container.

Place slide on the Thermocycler Adaptor with the active surface facing up and incubate 0h 1m 0s at 37°C .

DO NOT close the thermocycler lid. Maintain thermal cycler at 37°C .

Remove slide from Thermocycler Adaptor and if necessary, wipe excess liquid from the back of the slide, without touching the tissue sections.

Completely immerse the slide in the prechilled -20°C methanol.

Secure the tube cap to prevent methanol loss.

Incubate upright for 0h 30m 0s at -20°C .

Dispense the following volumes of Milli-Q water:

a. 500mL in Beaker 1

b. 800mL in Beaker 2

c. 800mL in Beaker 3

d. 800mL in Beaker 4

NOTE : Dispensed volume in each beaker can be used for two slides.

Prepare Eosin Mix.

DO NOT add pure eosin to tissue sections.

Remove slide from methanol and wipe excess liquid from the back of the slide, without touching the tissue sections.

Place on a flat, clean, nonabsorbent work surface.

Note : Some residual droplets may remain.

Add 500μL isopropanol to uniformly cover all tissue sections on the slide.

Incubate 0h 1m 0s at room temperature.

Tip : When incubating the slide with reagents, ensure that the slide is not in contact with any absorbent surface, like laboratory wipes, which may absorb the reagents.

Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.

Wipe excess liquid from the back of the slide, without touching the tissue sections.

Place on a flat, clean, nonabsorbent work surface.

Note : Some droplets may remain.

Air dry the slide for 0h 4m 0s.

Add 1mL Hematoxylin to uniformly cover all tissue sections on the slide.

Incubate 0h 5m 0s at room temperature.

Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.

Immerse the slide 5x in the water in Beaker 1.

Immerse the slide 15x in the water in Beaker 2.

Immerse the slide 15x in the water in Beaker 3.

Wipe excess liquid from the back of the slide without touching the tissue section.

Place on a flat, clean, nonabsorbent work surface.

Note : Some droplets may remain.

Add 1mL Bluing Buffer to uniformly cover all tissue sections.

Incubate 0h 2m 0s at room temperature.

Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe

Immerse the slide 5x in the water in Beaker 3.

Wipe excess liquid from the back of the slide without touching the tissue section.

Place on a flat, clean, nonabsorbent work surface.

Note : Some droplets may remain.

Add 1mL Eosin Mix to uniformly cover all tissue sections.

Incubate 0h 2m 0s at room temperature.

Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.

Immerse the slide 15x in the water in Beaker 4.

Wipe the back of the slide with a laboratory wipe.

Place on a flat, clean, nonabsorbent work surface and air dry until tissue is opaque.

Incubate slide on the Thermocycler Adaptor with the thermal cycler lid open for 0h 5m 0s at 37°C .

Proceed to tissue imaging.

Note : Ensure that the entirety of the tissue slice is in the same focal plane before imaging, to reduce the risk of stitching-induced image artifacts hindering downstream analyses.