TITLE: H&amp;E Staining for 10X Genomics Visium Imaging
AUTHORS: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill

[DESCRIPTION]
This protocol describes H&E staining of 10X Genomics Visium slides prior to imaging and is adapted from 10X Genomics protocol documented in “Methanol Fixation, H&E Staining & Imaging for Visium Spatial Protocols”. H&E staining and imaging is essential to ensuring target tissue/region/cells are mounted within the fiducial frame of the Visium slide.

[BEFORE_START]
Prepare Eosin mix fresh for each use. The tris-acetic acid buffer can be stored for up to 12 months.

[STEPS]
SECTION: Tissue Fixation
1. Prechill methanol (40mL/slide, dispensed in a 50-mL centrifuge tube) to -20 °C .

SECTION: Tissue Fixation
3. Remove slide from -80°C and place on dry ice in a sealed container.
SECTION: Tissue Fixation
4. Place slide on the Thermocycler Adaptor with the active surface facing up and incubate 1 min  at 37 °C . 

SECTION: Tissue Fixation
5. Remove slide from Thermocycler Adaptor and if necessary, wipe excess liquid from the back of the slide, without touching the tissue sections.
SECTION: Tissue Fixation
6. Completely immerse the slide in the prechilled -20 °C  methanol. 

SECTION: Tissue Fixation
7. Incubate upright for 30 min at -20 °C .

SECTION: Tissue Fixation
4.1. DO NOT close the thermocycler lid. Maintain thermal cycler at 37 °C .

SECTION: Tissue Fixation
6.1. Secure the tube cap to prevent methanol loss.
SECTION: Tissue Fixation
2. Place a Thermocycler Adaptor on a thermal cycler set at 37 °C  and equilibrate for 5 min . Heating the Thermocycler lid is not required.

SECTION: Tissue H&amp;E Staining
8. 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.
SECTION: Tissue H&amp;E Staining
9. Prepare Eosin Mix.

SECTION: Tissue H&amp;E Staining
10. Remove slide from methanol and wipe excess liquid from the back of the slide, without touching the tissue sections.
SECTION: Tissue H&amp;E Staining
11. Place on a flat, clean, nonabsorbent work surface.

Note: Some residual droplets may remain.
SECTION: Tissue H&amp;E Staining
12. Add 500μL isopropanol to uniformly cover all tissue sections on the slide.
SECTION: Tissue H&amp;E Staining
13. Incubate 1 min 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.
SECTION: Tissue H&amp;E Staining
14. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.
SECTION: Tissue H&amp;E Staining
15. Wipe excess liquid from the back of the slide, without touching the tissue sections.
SECTION: Tissue H&amp;E Staining
16. Place on a flat, clean, nonabsorbent work surface.

Note: Some droplets may remain.
SECTION: Tissue H&amp;E Staining
17. Air dry the slide for 4 min.

SECTION: Tissue H&amp;E Staining
18. Add 1mL Hematoxylin to uniformly cover all tissue sections on the slide.
SECTION: Tissue H&amp;E Staining
19. Incubate 5 min  at room temperature.

SECTION: Tissue H&amp;E Staining
20. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.
SECTION: Tissue H&amp;E Staining
21. Immerse the slide 5x in the water in Beaker 1.
SECTION: Tissue H&amp;E Staining
22. Immerse the slide 15x in the water in Beaker 2.
SECTION: Tissue H&amp;E Staining
23. Immerse the slide 15x in the water in Beaker 3.
SECTION: Tissue H&amp;E Staining
24. Wipe excess liquid from the back of the slide without touching the tissue section.
SECTION: Tissue H&amp;E Staining
25. Place on a flat, clean, nonabsorbent work surface.

Note: Some droplets may remain.
SECTION: Tissue H&amp;E Staining
26. Add 1mL Bluing Buffer to uniformly cover all tissue sections.
SECTION: Tissue H&amp;E Staining
27. Incubate 2 min  at room temperature.

SECTION: Tissue H&amp;E Staining
28. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe
SECTION: Tissue H&amp;E Staining
29. Immerse the slide 5x in the water in Beaker 3.
SECTION: Tissue H&amp;E Staining
30. Wipe excess liquid from the back of the slide without touching the tissue section.
SECTION: Tissue H&amp;E Staining
31. Place on a flat, clean, nonabsorbent work surface.

Note: Some droplets may remain.
SECTION: Tissue H&amp;E Staining
32. Add 1mL Eosin Mix to uniformly cover all tissue sections.
SECTION: Tissue H&amp;E Staining
33. Incubate 2 min  at room temperature.

SECTION: Tissue H&amp;E Staining
34. Discard reagent by draining and/or holding the slide at an angle with the bottom edge in contact with a laboratory wipe.
SECTION: Tissue H&amp;E Staining
35. Immerse the slide 15x in the water in Beaker 4.
SECTION: Tissue H&amp;E Staining
36. Wipe the back of the slide with a laboratory wipe.
SECTION: Tissue H&amp;E Staining
37. Place on a flat, clean, nonabsorbent work surface and air dry until tissue is opaque.
SECTION: Tissue H&amp;E Staining
38. Incubate slide on the Thermocycler Adaptor with the thermal cycler lid open for 5 min  at 37 °C .

SECTION: Tissue H&amp;E Staining
39. 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.
SECTION: Tissue H&amp;E Staining
9.1. DO NOT add pure eosin to tissue sections.