Oct 12, 2022
H&E Staining for 10X Genomics Visium Imaging
- Stephen Fisher1,
- Marielena Grijalva1,
- Rong Guo1,
- Sarah A Johnston1,
- Hieu Nguyen1,
- John Renz2,
- Jean G Rosario1,
- Steven Rudich2,
- Brian Gregory1,
- Junhyong Kim1,
- Kate O'Neill1
- 1University of Pennsylvania;
- 2Gift of Life Donor Program
Protocol Citation: Stephen Fisher, Marielena Grijalva, Rong Guo, Sarah A Johnston, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill 2022. H&E Staining for 10X Genomics Visium Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2owqqv1y/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: July 26, 2022
Last Modified: October 12, 2022
Protocol Integer ID: 67567
Funders Acknowledgements:
NIH
Grant ID: U54HD104392
Abstract
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.
Materials
Eosin Mix: 1mL
- 100µL Eosin Y solution (Sigma-Aldrich, HT110216-500mL)
- 900µL Tris-Acetic Acid Buffer (see below)
- Vortex to mix
- Prepare fresh for each use
Eosin Y solution aqueousMerckCatalog #HT110216-500ML
Tris-Acetic Acid Buffer: 200mL
- Dissolve 11g Tris base in 100mL nuclease-free water (Fisher, BP152-500)
- Adjust pH to 6.0 using 100% Acetic Acid (Fisher, A38-212)
- Bring volume to 200mL with nuclease-free water
- Filter through 0.2μm Corning 250mL Vacuum System
- Store at room temperature for up to 12 months
Tris BaseFisher ScientificCatalog #BP152
Acetic acid GlacialFisher ScientificCatalog #A38-212
Nuclease-free WaterContributed by users
Protocol:
- Tissue slice from OCT embedded tissue block, mounted on a 10X Visium slide
- Methanol suitable for HPLC ≥99.9%Merck Millipore SigmaCatalog #34860
- Mayer’s HematoxylinDakoCatalog #S3309
- Shandon™ Bluing ReagentThermo FisherCatalog #6769001
- Eosin Y solution aqueousMerckCatalog #HT110216-500ML
- Tris-Acetic Acid Buffer (see above)
- Eosin Mix (see above)
- Nuclease-free water or water filtered using a Milli-Q filtering systemAmbionCatalog #AM9932
- 10X Visium Thermocycler Adaptor (part of 10X Genomics Visium Accessory Kit; 1000194)
Visium Accessory Kit10x GenomicsCatalog #1000194
Equipment
Thermal cycler
NAME
T100 PCR thermal cycler
TYPE
BIO-RAD
BRAND
https://www.bio-rad.com/pt-br/product/t100-thermal
SKU
LINK
Before start
Prepare Eosin mix fresh for each use. The tris-acetic acid buffer can be stored for up to 12 months.
Tissue Fixation
Tissue Fixation
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 00:05:00 . Heating the Thermocycler lid is not required.
5m
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 00:01:00 at 37 °C .
1m
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 00:30:00 at -20 °C .
30m
Tissue H&E Staining
Tissue H&E Staining
19m
19m
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 00:01:00 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.
1m
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 00:04:00 .
4m
Add 1mL Hematoxylin to uniformly cover all tissue sections on the slide.
Incubate 00:05:00 at room temperature.
5m
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 00:02:00 at room temperature.
2m
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 00:02:00 at room temperature.
2m
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 00:05:00 at 37 °C .
5m
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.