CG000410 | Rev E

DEMONSTRATED PROTOCOL

Visium Spatial Gene Expression for FFPE –Deparaffinization, Decrosslinking, Immunofluorescence Staining & Imaging

Introduction
The Visium Spatial Gene Expression for FFPE is designed to measure mRNA in tissue sections derived
from formalin fixed & paraffin embedded (FFPE) tissue samples and requires a Visium Spatial slidewith intact tissue sections as input. Immunostaining tissue sections with fluorescently labeled
antibodies enables simultaneous protein detection. This protocol outlines deparaffinization,
decrosslinking, immunofluorescence (IF) staining, and imaging of tissue for use with 10x Genomics
Visium Spatial Gene Expression for FFPE assay. Deparaffinized, decrosslinked, and stained tissue
sections are inputs for the downstream Visium Spatial Gene Expression for FFPE workflow.

Additional GuidanceConsult the Visium Spatial Gene Expression for FFPE - Tissue Preparation Guide(Document CG000408) for complete information on sectioning FFPE tissue blocks andplacing sections on Visium Spatial slides. Ensure that tissue sections have been placed
onto the appropriate slide before starting this Demonstrated Protocol. Consult the
Visium Spatial Gene Expression for FFPE Imaging Guidelines (Document CG000436) toverify imaging settings before starting this Demonstrated Protocol. After completing
this Demonstrated Protocol (CG000410), proceed with the Visium Spatial Gene
Expression for FFPE - User Guide (CG000407).

Contents

2		 Reagent Kits
3		 Recommended Thermal Cyclers

1.4 Immunofluorescence Staining

30 2. Tissue Imaging

4		 Specific Reagents & Consumables

30
31
31

5		 Tips & Best Practices

32 Troubleshooting

16 Protocol Steps & Timing

32 Results

3		 Workflow Overview

17		 1. Deparaffinization, Decrosslinking &		 IF Staining
17
18
21
23

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1.1 Preparation - Buffers
Protocol Overview
1.2 Deparaffinization
1.3 Decrosslinking

2.1 Imaging System Recommendations
2.2 Imaging
2.3 Coverslip Removal

Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

Reagent Kits

CG000410 | Rev E

Visium Spatial Gene Expression for FFPE Reagent Kits
Refer to SDS for handling and disposal information

Visium Spatial Gene
Expression Slide Kit,
16 rxns
PN-1000185

Visium Spatial Gene
Expression Slide Kit,
4 rxns
PN-1000188

VisiumSpatial Gene Expression Slide Kit
16 rxns, PN-1000185
store at ambient temperature or at –20°C according to kit label#
PN
Visium Spatial Gene Expression Slide
42000233*Visium Slide Seals, 40-pack1
2000284
Visium Cassette & Gasket Assembly, 4-pack 1
2000282

VisiumSpatial Gene Expression Slide Kit
4 rxns, PN-1000188
store at ambient temperature or at –20°C according to kit label#
PN
Visium Spatial Gene Expression Slide1
2000233*Visium Slide Seals, 12-pack1
2000283
Visium Cassette & Gasket Assembly, 1-pack 1
2000281*Visium Slide Seals may come in varying dimensions and quantities in different lots. Check thenumber of slide seals in the kit. Additional seals may be required. Refer to page 13 (Visium Slide
Seal Application & Removal) of this Demonstrated Protocol for instructions on how to resize
seals or cut additional seals...

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

10x Genomics Accessories
Product

Part Number (Kit)

Thermocycler Adaptor

Part Number (Item)
3000380

Visium Spatial Imaging Test Slide
10x Magnetic Separator

1000194

Slide Alignment Tool

2000235
2300033000433

Recommended Thermal Cyclers
Supplier
Bio-RadEppendorf
Thermo Fisher Scientific

Description
C1000 Touch Thermal Cycler with
96-Deep Well Reaction Module
MasterCycler Pro(discontinued)
Veriti 96-Well Thermal Cycler

Part Number
1851197
North America 950030010
International 6321 000.019
4375786

Workflow Overview
Tissue Preparation Guide
Section tissue onto slides.
Demonstrated Protocol CG000408

Imaging Guidelines
Optimize imaging settings for Visium Spatial
Gene Expression for FFPE workflow.
Technical Note CG000436

Deparaffinization + Decrosslinking +IF Staining
Deparaffinize, decrosslink, stain, and image tissue.
Demonstrated Protocol CG000410

Library Construction
Construct Visium Spatial Gene Expression- FFPE libraries.
User Guide CG000407
Visit the 10x Genomics Support website for the most current documentation.

10xgenomics.com

3Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Specific Reagents & Consumables
For each item, a number of vendor options are listed. Choose item based on availability and preference.
Item

Alternatives/Options

VendorPart Number

XyleneXylene, Reagent Grade
Xylene, Histological Grade
Ethyl Alcohol, 200 Proof
Ethanol absolute ≥99.5%, TechniSolv, pure (Europe Only)
TE Buffer (pH 9.0)
Alternatively, prepare TE buffer using Tris and EDTA andadjust the pH to 9.0
Tris Base
For preparing TE buffer (pH 9.0), alternative to Genemed
product
UltraPure 0.5 M EDTA, pH 8.0
For preparing TE buffer (pH 9.0), alternative to Genemed
product
Dilute to 1.0 M HCl stock
For preparing TE Buffer (pH 9.0), alternative to Genemed
product
PBS - Phosphate Buffered Saline (10X) pH 7.4, RNase-freeMillipore Sigma
Millipore Sigma
Millipore Sigma
VWR
GeneMed214736534056
E7023
83813.360DP
10-0046

Fisher Scientific

BP152-500

Thermo Fisher Scientific15575020

Millipore Sigma258148

Thermo Fisher ScientificAM9624Albumin, Bovine Serum, 10% Aqueous Solution, NucleaseFree
Tween 20 Surfact-Amps Detergent Solution (10% solution)

Millipore Sigma

126615-25ML

Thermo Fisher Scientific28320

Nuclease-free
water
DAPI
Antibody

Nuclease-free Water (not DEPC-Treated)

Thermo Fisher ScientificAM9937DAPI Solution (1 mg/ml)-

Thermo Fisher Scientific
-

62248
-

RNase
inhibitor
Mounting
mediumSection dryer
ovenProtector RNase Inhibitor

Millipore Sigma

3335399001

SlowFade Diamond Antifade Mountant

Thermo Fisher ScientificS36967

Epredia High Capacity Section Dryer

Fisher Scientific

A84600051

Slide holders
Coverslips

Slide Holders, 24-place
Fisherbrand Cover Glasses: Rectangles

VWR
Fisher Scientific25608-868
12-544-EP

Cover Glasses, Rectangular

VWR

16004-322

Staining jar/dishes

Coplin Jar

VWR

100500-232

Staining Dishes

VWR

25608-906

Sealing film

Microseal ‘B’ PCR Plate Sealing Film, adhesive

Bio-Rad

MSB1001

Beakers--

Ultrapure/Milli-Q Water,from Milli-Q Integral Ultrapure Water System or equivalent--

Ethanol
TE Buffer(pH 9.0)
Or
Prepare using
Tris and 0.5 M
EDTA

PBS
BSA
10% Tween-20

Additional Materials

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4Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Tips & Best Practices

Tips & Best Practices section
includes additional guidance

Signifies critical steprequiring accurate execution

Troubleshooting section
includes additional guidance

General Reagent Handling• Thoroughly mix reagents before use.
• Use a pH meter to adjust pH as necessary during buffer preparation.

Pipette Calibration• Follow manufacturer’s calibration and maintenance schedules.

Visium Spatial Gene Expression Slide• Visium slides include 4 Capture Areas (6.5 x 6.5 mm), each with ~5,000 uniquegene expression spots.
• Each gene expression spot includes primers with a unique Spatial Barcode.
• The active surface of the slide is defined by a readable label that includes theserial number.
• The tissue sections are always placed on the active surface of the Capture Areas.
For more information, consult the Visium Spatial Gene Expression for FFPE –Tissue Preparation Guide (Demonstrated Protocol CG000408).
• Always store slides in a cool, dry environment. After tissue placement, store theslides at room temperature in a low moisture environment such as a desiccator.

Visium Spatial Gene Expression Slide
Label onActiveSurface(with serial
number)Capture Areas(place tissue sections)
Note the serial number on the slide label; will be required for downstream analysis.

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5Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Slide Handling• Always wear gloves when handling slides.
• Ensure that the active surface of a slide faces up and is never touched. The orientation of the label on the slide defines the active surface.
• The tissue sections should always be on the active surface of the slide. DO NOT
touch the tissue sections on the slide.
• Minimize exposure of the slides to sources of particles and fibers.
• When immersing slides in deparaffinization solutions and water, ensure that thetissue sections are completely submerged.
• Xylene and ethanol may cause the slide label to come off. Keep the label above thesurface of the liquid when immersing in xylene and ethanol.
• Keep the slide flat on the bench when adding reagents to the active surface.
• Ensure that no absorbent surface is in contact with the reagents on the slide
during incubation.

Active Surface with Tissue Sections

Immersing Slide
Correct

Incorrect
Not
immersed

Reagent on Slide
Correct

Incorrect

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6Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Slide Incubation Guidance
Incubation at a specified temperature
Incubation using a Section Dryer Oven:• Place the slides in a slide drying rack.
• Close the lid when incubating the slide in the oven.

Place in a Rack

Slides in images
are representative.

Incubation using a Thermal Cycler:• Position a Thermocycler Adaptor on a thermal cycler that is set at the incubation
temperature.
• Ensure that the Thermocycler Adaptor is in contact with the thermal cycler surface uniformly.
• When incubating a slide, position the slide on the Thermocycler Adaptor with theactive surface facing up.
• Ensure that the entire bottom surface of the slide is in contact with Thermocycler
Adaptor.
• When incubating a slide encased in a Visium Cassette, place the assembled unit
on the Thermocycler Adaptor with the wells facing up. The Visium Cassetteshould always be sealed when in the Thermocycler Adaptor.
Place Thermocycler Adaptor

Incubate Slide

Incubate Assembled Visium Cassette

DO NOT close lid

Close lid
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7Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Incubation at room temperature• Place the slide/Visium Cassette on a flat, clean, non-absorbent work surface.
• Ensure that no absorbent surface is in contact with the reagents on the slide
during incubation.

Visium Cassette• The Visium Cassette encases the slide and creates leakproof wells for adding reagents.
• Place the slides in the Visium Cassette only when specified.
• The Visium Cassette includes a removable Visium Gasket.
• An Insert Clip and four tabs at the back of the Visium Cassette are used forholding the slide in the cassette, as shown.
• The removable Visium Gasket corresponds to the Capture Areas on the slides.
• The Visium Cassette may be assembled using the Slide Alignment Tool ormanually. Instructions for both are provided in the following section.
• See Visium Cassette Assembly & Removal instructions for details.
• Ensure that the back of the Visium Cassette is facing the user before assembly.
The active surface of the slide with tissue sections will face down such that theslide label is no longer readable.
• Practice assembly with a plain glass slide (75 x 25 x 1 mm).
• Applying excessive force to the slide may cause the slide to break.

Visium Cassette

Slide Alignment Tool

Back

Front

Tab 3
Insert
Clip
Tab 4

Tab 1
VisiumGasketTab 2

Ridges10xgenomics.com

8Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

Visium Cassette
Assembly

CG000410 | Rev E

Position Visium Cassette along alignment
tool ridges

Push Insert Clip along the ridge & press
Visium Cassette down

1

2

Exercise caution whenhandling slide edges toprevent injury.

InsertClip

Ridge
InsertClip

Visium Cassette secured onalignment tool3

Position Visium Gasket to align with
Visium Cassette cutouts

4 4

Insert long edge of slide undertabs 1 & 2; ensure slide is flush

5 5

Press
down
Press
down

Slide Slide Tab 1
Tab 2
label label
Tab 1

Visium Gasket
GasketPress slide down until it is flush with the
Visium Gasket and under tabs 3 & 4

6
Press down

Slide insertion may push
Visium Gasket out ofalignment with slide
cutouts. Adjust if necessary.
Tab 3

Tab 4

Active surface faces
Visium Gasket
Remove Visium Cassette while pressing slide
against the Visium Gasket

7

Pull upSlides inimages are
representative.
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9Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

Visium Cassette
RemovalPosition Visium Cassette alongalignment tool ridges

1

Push Insert Clip along the ridge& press down2

InsertClip

Ridge

Press down

Visium Cassette sits
securely on alignment tool3

Slides inimages are
representative.

CG000410 | Rev E

Lift slide at Visium
Cassette groove

4

Groove10xgenomics.com

10Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Manual Visium Cassette Assembly & Removal
Assemblya. Remove the Visium Gasket from the Visium Cassette and re-insert the Visium
Gasket, ensuring that the Visium Gasket and Visium Cassette cutouts are aligned.
b. Align the label on top of the slide to the top of the Visium Cassette, as shown.
c. Insert the slide under tabs 1 and 2. Ensure that the long edge of the slide is flushwith the side of the Visium Cassette.
d. Press the insert clip very firmly by applying even force on the lower
part of the insert clip.
e. Place a finger in between tab 3 and the top of the cassette, and one finger between
tab 4 and the bottom of the cassette.
Press down on the slide evenly until the slide is under each tab and release theinsert clip.

Removala. Press the insert clip very firmly to release the slide from the cassette.
b. Lift slide at Visium Cassette groove between tabs 3 and 4 until the slide can beremoved.
Visium Cassette

Visium Cassette Assembly
Squeeze
Insert
Clip

Back

Front

Tab 3

Tab 1

InsertClip

VisiumGasket

Tab 4

Tab 2

Label facing
down

Slide
under tabs
Insert Clip - Press Firmly
Lift
here

Press
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11Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Reagent Addition to Wells• Place the assembled slide in the Visium Cassette flat on a clean work surface.
• Dispense reagents along the side of the wells without touching the tissue sections
and without introducing bubbles.
• Always cover the tissue section completely when adding reagents to the well. A
gentle tap may help spread the reagent more evenly.
• Ensure that no bubbles are introduced in the process.
Reagent Addition/Removal
DO NOT
introduce
air bubbles

Add/remove along
the side of wells

Add reagent along the slide of the wells

Reagent Removal from Wells• Place the assembled slide in the Visium Cassette flat on a clean work surface.
• Slightly tilt the Visium Cassette while removing the reagent.
• Place the pipette tip to the bottom edge of the wells.
• Remove reagents along the side of the wells without touching the tissue
sections and without introducing bubbles.
• Ensure that no bubbles are introduced in the process.
• Remove all liquid from the wells in each step. To ensure complete removal, checkthe bottom of the well by tilting the cassette slightly. A meniscus at the bottom of
the well will indicate the presence of liquid in the well.

Place the pipette tip to the bottom edgeof the wells

Meniscus at the bottom indicates liquid.
DO NOT leave liquid behind

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12Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Visium Slide Seal Application & Removal
To generate new or resize Visium Slide Seals, use one of the provided seals (PN2000283/2000284) as a template to cut additional seals from MicroSeal 'B' PCR Plate
Sealing Film (PN-MSB1001; listed in Specific Reagents & Consumables). Contact
support@10xgenomics.com if assistance is required.

Application• Place the Visium Cassette flat on a clean work surface.
• Remove the back of the adhesive Visium Slide Seal.
• Align the Visium Slide Seal with the surface of the Visium Cassette and apply
while firmly holding the Visium Cassette with one hand.
• Press on the Visium Slide Seal to ensure uniform adhesion.

Removal• Place the Visium Cassette flat on a clean work surface.
• Pull on the Visium Slide Seal from the edge while firmly holding the Visium Cassette.
• Ensure that no liquid splashes out of the wells.

Visium Slide Seal Application

Tissue Detachment on Visium Slides• Monitor section adhesion on the Visium slides throughout the workflow. For
more information, consult the Visium Spatial Gene Expression for FFPE – Tissue
Preparation Guide (Demonstrated Protocol CG000408).
• Tissue detachment during the workflow can impact performance.

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Antibody Optimization
Prior testing of the antibodies is recommended on the same tissue before performing
immunofluorescence staining in combination with the Visium Spatial Gene Expression for FFPE workflow. Determination of the optimal antibody concentration is
crucial for executing this protocol.
• Optimal antibody concentrations for this Demonstrated Protocol may differ fromother applications. Composition of reagents and buffers may also differ fromother immunofluorescence applications.
• To optimize antibody concentration, draw representative frames on the backof a 75 x 25 x 1 mm plain glass slide using the example slide layout. Place tissue
sections in the frames on the front of the slide for compatibility with the Visium
Cassette. Ensure that tissue sections used during optimization are similar in size
to tissue sections used for Visium Spatial Gene Expression for FFPE workflow.
• Execute the Deparaffinization, Decrosslinking & Immunofluorescence Staining
protocol using a range of antibody concentrations. A starting concentration of
0.01 μg/μl (0.5 μg/sample) is recommended.
• Select the antibody concentration that results in the specific staining of desired
cells, while minimizing nonspecific background staining.
• When optimizing the antibody, ensure that stained slides can be imaged according to the imaging guidelines listed in Visium Spatial Gene Expression for FFPE
Imaging Guidelines Technical Note (CG000436).
• Wash Visium Cassette and Visium Gasket after immunofluorescence staining.
See Visium Cassette & Visium Gasket Cleaning for more information.

28.5 mm

Example Slide
Layout – Gene
Expression Slide

8 mm

8 mm

11.25 mm

1.3 mm

3.5 mm10xgenomics.com

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Visium Cassette and Visium Gasket Cleaning
Mark the top portion of the Visium
Gasket that faced the slide with an
alcohol resistant marker

Remove slide from Visium Cassette12

Remove Visium Gasket

Rinse Visium Cassette and Gasket
with ultrapure water

44

33

GrooveSpray with 70% isopropanol, then rinse withultrapure water

4
5

Spray with 70% isopropanol a second time, thenrinse with ultrapure water46

GrooveGrooveAir dry37

Re-insert the Visium Gasket such that the marked
portion is at the top of the Visium Cassette and now
faces the Visium Cassette.

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Protocol Steps& Timing~5.5 hours
Steps

TimingStop & Store

Step 1 – Deparaffinization
1.2

Deparaffinization

60 minStep 2 – Decrosslinking
1.3

Decrosslinking

60 minStep 3 – Immunofluorescence Staining
1.4

Immunofluorescence Staining
60-150 min depending upon the type of immunofluorescence staining
performed, see below.

60 min1.4A Fluorophore Conjugated Primary Antibodies
OR
1.4B Primary and Secondary Antibodies

150 min

Step 4 – Tissue Imaging
2.2
2.3

Imaging
Coverslip Removal

variable
5 min STOP 4°C ≤2 weeks

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

1. Deparaffinization, Decrosslinking &Immunofluorescence (IF) Staining
1.0 Overview
This chapter provides guidance on deparaffinization, decrosslinking, andimmunofluorescence (IF) staining of Visium slides containing FFPE tissue sections thatare dried overnight in a desiccator. Ensure that microscope settings have been verified
and imaging programs have been created before starting this program. Consult the
Visium Spatial Gene Expression for FFPE Imaging Guidelines Technical Note (CG000436)
for more information. If staining using fluorophore conjugated primary antibodies,
proceed directly to step 1.4A (Immunofluorescence staining- Fluorophore ConjugatedPrimary Antibodies) after completing step 1.3 (Decrosslinking). If staining using primaryand secondary antibodies, proceed directly to step 1.4B (Immunofluorescence stainingPrimary and Secondary Antibodies) after completing step 1.3 (Decrosslinking).
Deparaffinization
Decrosslinking
IF Staining
Coverslipping

1.1 Preparation - Buffers
For Deparaffinization
Items
Xylene100% Ethanol
96% Ethanol
70% Ethanol
Milli-Q Water

Preparation & Handling
Label two coplin jars as Xylene Jar 1 and 2. Dispense 30 ml xylene in each.
Label three coplin jars as 100% Ethanol Jar 1, 2, and 3. Dispense 30 ml
100% ethanol in each.
Label two coplin jars as 96% Ethanol Jar 1 and 2. Dispense 30 ml 96%
ethanol in each.
Label one coplin jar as 70% Ethanol Jar. Dispense 30 ml 70% ethanol.
Label one coplin jar as Milli-Q Water Jar. Dispense 30 ml Milli-Q water.
Alternatively, use a 50-ml centrifuge tube or a beaker for each individual
slide. Clean beaker with Milli-Q Water then RNase decontamination
solution in between use.

Alternatively, a slide staining dish can also be used in place of a coplin jar. Adjust the volumes ofdeparaffinization solutions and water, accordingly. Use xylene-resistant dishes, for immersion in xylene.
Use xylene-resistant gloves or forceps for deparaffinization. Prepare fresh reagents every week. Use
fresh reagents after every 20 slides or every month (whichever comes first).

For Decrosslinking
Items
1X PBSTE Buffer, pH
9.0 OrPrepare TE
Buffer, pH 9.0

				Preparation & Handling
Prepare 1X PBS using nuclease-free water.

TE Buffer Preparation (Prepare fresh and maintain at room temperature)• Dissolve 1.21 g Tris base in 950 ml nuclease-free water.
• Adjust the pH to 9.0 with 1.0 M HCl.
• Add 2 ml of 0.5 M EDTA and adjust the pH to 9.0 using 1.0 M HCl.
Bring the volume to 1000 ml using nuclease-free water.
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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Protocol Overview
Deparaffinization		

Incubate at
60°C
for 2 hImmerse in
96% ethanol& incubate
for 3 min

96% Ethanol
Jar 1
Immerse in
96% ethanol& incubate
for 3 min

96% Ethanol
Jar 1

Immerse in
85% ethanol& incubate
for 3 min

~3 h

Thermal cycler* oroven

100% Ethanol
Jar 3

85% Ethanol
Jar

Immerse inxylene & incubate
for 10 min

Immerse inxylene & incubate
for 10 min

Xylene Jar 1

Immerse in
100% ethanol& incubate
for 3 min

Immerse in
70% ethanol& incubate
for 3 minXylene Jar 2
Immerse in
100% ethanol& incubate
for 3 min

Immerse in
100% ethanol& incubate
for 3 min

100% Ethanol
Jar 2

Immerse inmilli-Q water& incubate
for 20 sec

70% Ethanol
Jar

100% Ethanol
Jar 1Milli-Q
Water* Keep the thermal cycler lid open during incubation, allow slide to cool
to room temperature and immerse in xylene after 60°C heating step

Decrosslinking~1 hIncubate at 37°C
for 3 minDeparaffinized
slide

Place slide in
Visium Cassette
Thermal cycler(Keep the lid
open)

Add TE buffer,
pH 9.0(150 �l/well)Deparaffinizedslide in the
Visium Cassette

Proceed to appropriate
Immunofluorescence Staining

RemovePBS

RemoveTE Buffer

Add PBS(100 �l/well) &Incubate atroom temperaturefor 5 min Remove

Add TE buffer,
pH 9.0(100 �l/well)
Apply Slide
Seal &
Incubate at
70°C
for 1 hRemovefrom thermal
cycler
Thermal cycler

TE Buffer
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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Immunofluorescence Staining – Fluorophore Conjugated Primary Antibodies

Add Antibody Solution(100 �l/well) &Incubate at room
temperaturefor 60 min

Add 1X Blocking Buffer(100 �l/well) &Incubate at room
temperature for 5 min
Decrosslinked
slide in the
Visium Cassette~1 hRemoveBlocking
BufferRemoveAntibody
Solution

Add 1X Blocking
Buffer(100 �l/well) &Incubate at room
temperaturefor 1 minRemoveBlocking
BufferRepeat 3x
Remove from
PBS & proceed
to Coverslipping

Immerse20 times in
1X PBS

Remove fromthe Visium
Cassette

1X PBS

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19Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

Immunofluorescence Staining – Primary and Secondary Antibodies

Add 1X Blocking Buffer(100 �l/well) &Incubate at room
temperature for 5 min
Decrosslinked
slide in the
Visium Cassette

RemoveBlocking
BufferCG000410 | Rev E~2.5 h

Add Primary Antibody
Solution(100 �l/well) &Incubate at room
temperaturefor 60 min
RemoveAntibody
Solution

Add 1X Washing
Buffer(100 �l/well) &Incubate at room
temperaturefor 5 minRemoveWashing
BufferRepeat 2x

Add Secondary
Antibody Solution(100 �l/well) &Incubate at room
temperaturefor 60 min
RemoveAntibody
Solution

Add 1X Washing
Buffer(100 �l/well) &Incubate at room
temperaturefor 5 minRemoveWashing
BufferRepeat 2x

Remove from
PBS & proceed
to Coverslipping

Immerse20 times in
1X PBS

Remove fromthe Visium
Cassette

1X PBS
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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

1.2 Deparaffinization
Deparaffinization steps should be performed in a fume hood due to the hazardous
nature of xylene.
Visium Spatial slide contains a readable label with a serial number on the active
surface of the slide. The label may come off during deparaffinization steps. In suchcases, the serial number etched on the slide can be used.
a. Retrieve the slide with tissue sections from the desiccator after overnight drying.
b. Place slides in a Section Dryer Oven and incubate uncovered at 60°C for 2 h. Keep
the oven lid closed during incubation.

Incubation in a Section Dryer Oven

Place slide in a rack

Place slide sideways and keep theoven door closed during incubation

Alternatively, place a Thermocycler Adaptor on a thermal cycler set at 60°C. Place
slide on the Thermocycler Adaptor with the active surface facing up and incubate
2 h at 60°C. DO NOT close the thermal cycler lid.

Incubation in a Thermal Cycler
DO NOT close
the lid

Place Thermocycler Adaptor

Incubate Slide for 2 h at 60oC

c. Remove from the oven or thermal cycler and allow the slide to cool down to room
temperature.

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

d. Gently immerse the slide in the xylene in
Xylene Jar 1. Secure the jar cap to prevent
xylene loss.

Submerge in Xylene/Ethanol

When immersing slides in xylene, ensure thatthe tissue sections are completely submerged
and the xylene does not reach the label.
e. Incubate for 10 min.
f.

CG000410 | Rev E

Label above thesurface of theliquidSections fully
submerged

During incubation, set up a thermal cycler to
37°C. Place a Thermocycler Adaptor on thethermal cycler.

g. Gently immerse slide in the xylene in Xylene
Jar 2 and incubate for 10 min.
h. Gently immerse slide in the 100% Ethanol Jar 1 for 3 min.
When immersing slides in ethanol, ensure that the tissue sections are completely
submerged and the ethanol does not reach the label.
i.

Gently immerse slide in the 100% Ethanol Jar 2 for 3 min.

j.

Gently immerse slide in the 100% Ethanol Jar 3 for 3 min.

k. Gently immerse slide in the 96% Ethanol Jar 1 for 3 min.
l.

Gently immerse slide in the 96% Ethanol Jar 2 for 3 min.

m. Gently immerse slide in the 85% Ethanol Jar for 3 min.
n. Gently immerse slide in the 70% Ethanol Jar for 3 min.
o. Gently immerse slide in the water in the tube/beaker and incubate for 20 sec.
p. Let the slide air dry and proceed to Decrosslinking.

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CG000410 | Rev E

1.3 Decrosslinking
Reagent addition and removal should be done carefully. Remove reagents along theside of the wells without touching the tissue sections and without introducing
bubbles.
a. Place a Thermocycler Adaptor on a thermal cycler set at 37°C. Place slide on the
Thermocycler Adaptor with the active surface facing up and incubate 3 min at
37°C. DO NOT close the thermal cycler lid.
b. Prepare the thermal cycler with the following incubation protocol and start the
program.
Lid Temperature

Reaction Volume

Run Time

70°C

-

1h

Step

TemperatureTime

Pre-equilibrate

70°C

Hold

Decrosslinking

70°C

00:60:00

Hold

22°C

Hold

c. Place the slide in the Visium Cassette.
See Tips & Best Practices for assembly instructions.
Practice assembly with a blank slide.
d. Add 150 µl TE Buffer (pH 9.0) along the side of the wells.
e. Remove all TE Buffer from the wells.
f.

Add 100 µl TE Buffer (pH 9.0) along the side of the wells.

g. Apply Visium Slide Seal on the Visium Cassette and place the cassette on the
Thermocycler Adaptor at 70°C.
h. Close the thermal cycler lid. Skip pre-equilibrate step and initiate Decrosslinking.
i.

After decrosslinking is complete, remove the cassette from the Thermocycler
Adaptor and place on a flat, clean work surface.

j.

Remove the slide seal and using a pipette, remove all TE Buffer from the wellcorners.

k. Add 100 µl 1X PBS along the side of the wells to uniformly cover the tissue
sections, without introducing bubbles.
l.

Incubate for 5 min at room temperature.

m. Remove all PBS from the wells.
n. Proceed immediately to appropriate Immunofluorescence Staining.

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CG000410 | Rev E

1.4 Immunofluorescence Staining
Choose appropriate staining protocol depending upon the antibodies used

1.4A Immunofluorescence Staining – Fluorophore Conjugated Primary Antibodies
a. Prepare 2X Blocking Buffer on ice. Pipette mix 10x and centrifuge briefly.

2X Blocking Buffer
If using primraryand secondary
antibody, proceed
directly to 1.4B.

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%
(µl)

PBS

10X

2X

80.0

352.0

704.0

BSA

10%

4%

160.0

704.0

1408.0Tween-20

10%

0.2%

8.0

35.2

70.4

40 U/�l

2 U/�l

20.0

88.0

176.0

Add reagents in the order
listed. Maintain on ice

Protector RNase
Inhibitor
Nuclease-free Water

-

132.0

580.8

1161.6

Total

-

400.0

1760.03520.0b. Prepare Antibody Solution on ice. Pipette mix 10x and centrifuge briefly.

Antibody Solution
Add reagents in the order
listed. Maintain on ice

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%(µl)

2X Blocking Buffer

2X

1X

50.0

220.0

440.0

Nuclease-free Water

Variable

Variable

Variable

Variable

Variable

Diluted DAPI
Dilute 1:100 innuclease-free water
before using.

10 �g/ml

0.2 �g/ml

2.0

8.8

17.6

Antibody #1

Variable

Variable

Variable

Variable

Variable

Antibody #2(Optional)

Variable

Variable

Variable

Variable

Variable

Antibody #3(Optional)

Variable

Variable

Variable

Variable

Variable

-

-

100.0

440.0

880.0

Total

*Antibody dilution can change depending on the antibody, ranging from 1:50 up to 1:1000. Antibody
volumes will depend on concentrations determined during antibody optimization. Add an appropriate
volume of nuclease-free water based on the amount of added antibody to achieve the stated total volume.
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24Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

c. Prepare 1X Blocking Buffer on ice. Pipette mix 10x and centrifuge briefly.

1X Blocking Buffer
Add reagents in the order
listed. Maintain on ice

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%(µl)

2X Blocking Buffer

2X

1X

250.0

1100.02200.0

Nuclease-free Water

-

-

250.0

1100.02200.0

Total

-

-

500.0

2200.04400.0d. Add 100 µl 1X Blocking Buffer along the side of the wells.
e. Incubate for 5 min at room temperature.
f.

Remove all Blocking Buffer from the wells.

g. Add 100 µl Antibody Solution along the side of the wells. Tap gently to ensure
uniform coverage.
h. Incubate for 60 min at room temperature.
i.

Remove Antibody Solutionj.

Add 100 µl 1X Blocking Buffer along the side of the wells.

k. Incubate for 1 min at room temperature.
l.

Remove all Blocking Buffer from the wells.

m. Repeat j-l three more times for a total of four washes.
n. Remove the slide from Visium Cassette.
See Tips & Best Practices for removal instructions.
o. Gently immerse slide 20 times in 1X PBS in 50-ml centrifuge tube.
p. Remove slide from the PBS and proceed immediately to Coverslipping.
DO NOT let the slide dry.

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

1.4B Immunofluorescence Staining – Primary and Secondary Antibodies
a. Prepare 2X Blocking Buffer on ice. Pipette mix 10x and centrifuge briefly.

2X Blocking Buffer

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%
(µl)

PBS

10X

2X

80.0

352.0

704.0

BSA

10%

4%

160.0

704.0

1408.0Tween-20

10%

0.2%

8.0

35.2

70.4

40 U/�l

2 U/�l

20.0

88.0

176.0

Add reagents in the order
listed. Maintain on ice

Protector RNase
Inhibitor
Nuclease-free Water

-

132.0

580.8

1161.6

Total

-

400.0

1760.03520.0b. Prepare 1X Blocking Buffer on ice. Pipette mix 10x and centrifuge briefly.

1X Blocking Buffer
Add reagents in the order
listed. Maintain on ice

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%(µl)

2X Blocking Buffer

2X

1X

250.0

1100.02200.0

Nuclease-free Water

-

-

250.0

1100.02200.0

Total

-

-

500.0

2200.04400.0c. Prepare Primary Antibody Solution on ice. Pipette mix 10x and centrifuge briefly.

Primary Antibody
Solution

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%
(µl)

2X

1X

50.0

220.0

440.0

Antibody #1

Variable

Variable

Variable

Variable

Variable

Antibody #2(Optional)

Variable

Variable

Variable

Variable

Variable

Antibody #3(Optional)

Variable

Variable

Variable

Variable

Variable

-

-

100.0

440.0

880.0

Add reagents in the order
listed. Maintain on ice

2X Blocking Buffer

Total*Antibody dilution can change depending on the antibody, ranging from 1:50 up to 1:1000. Antibody
volumes will depend on concentrations determined during antibody optimization.

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

d. Prepare 1X Washing Buffer. Pipette mix 10x and centrifuge briefly.

1X Washing Buffer

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%
(µl)

PBS

10X

1X

100.0

440.0

880.0

Tween-20

10%

0.1%

10.0

44.0

88.0

Nuclease-free Water

-

890.0

3916.07832.0Total-

1000.04400.08800.0

Add reagents in the order
listed. Maintain on ice

e. Prepare Secondary Antibody Solution on ice. Pipette mix 10x and centrifuge
briefly.

Secondary Antibody
Solution

Stock

Final

1X
(µl)

4X+ 10%(µl)

8X + 10%(µl)

2X Blocking Buffer

2X

1X

50.0

220.0

440.0

Diluted DAPI
Dilute 1:100 innuclease-free water
before using.

10 �g/ml

0.2 �g/ml

2.0

8.8

17.6

Antibody #1

Variable

Variable

Variable

Variable

Variable

Antibody #2(Optional)

Variable

Variable

Variable

Variable

Variable

Antibody #3(Optional)

Variable

Variable

Variable

Variable

Variable

-

-

100.0

440.0

880.0

Add reagents in the order
listed. Maintain on ice

Total*Antibody dilution can change depending on the antibody, ranging from 1:50 up to 1:1000. Antibody
volumes will depend on concentrations determined during antibody optimization.

f.

Add 100 µl 1X Blocking Buffer along the side of the wells.

g. Incubate for 5 min at room temperature.
h. Remove all Blocking Buffer from the wells.
i.

Add 100 µl Primary Antibody Solution along the side of the wells. Tap gently toensure uniform coverage.

j.

Incubate for 1h at room temperature.

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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

k. Remove Primary Antibody Solution.
l.

Add 100 µl 1X Washing Buffer along the side of the wells.

m. Incubate for 5 min at room temperature.
n. Remove 1X Washing Buffer from the wells.
o. Repeat l-n two more times for a total of three washes.
p. Add 100 µl Secondary Antibody Solution along the side of thewells. Tap gently to ensure uniform coverage.
q. Incubate for 60 min at room temperature.
r.

Remove Secondary Antibody Solution.

s. Add 100 µl 1X Washing Buffer along the side of the wells.
t.

Incubate for 5 min at room temperature.

u. Remove 1X Washing Buffer from the wells.
v. Repeat s-u two more times for a total of three washes.
w. Remove the slide from Visium Cassette.
See Tips & Best Practices for removal instructions.
x. Gently immerse slide 20 times in 1X PBS in 50-ml centrifuge tube.
y. Remove slide from the PBS and proceed immediately to
Coverslipping.
DO NOT let the slide dry.

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CG000410 | Rev E

1.5 Coverslippinga. Place slide on a flat, clean, non-absorbent work surface. Some residual droplets
may remain.
b. Add 5 drops of SlowFade Diamond Antifade Mountant to uniformly cover all
tissue sections on the slide.
c. Apply the coverslip at an angle on one end of the slide. Slowly lower the coverslip,
without introducing bubbles. Allow the mounting medium to spread and settle.
d. If needed, remove any large excess of mounting media by carefully wicking awayfrom the edge of the coverslip with a laboratory wipe. Be careful not to movecoverslip and disturb the tissue.
e. Once coverslipping is complete, immediately proceed with imaging.
DO NOT let the attached coverslip dry.
DO NOT use Cytoseal or nail polish for securing the coverslip.

Cover uniformly withmounting medium

Apply coverslip

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CG000410 | Rev E

2. Tissue Imaging
2.0 Overview
This chapter provides guidance on imaging Visium slides containing immunofluorescent
stained FFPE sections and coverslip removal.

Fluorescent Imaging & Coverslip Removal
Proceed to Visium
Spatial Gene
Expression for FFPE
User Guide

Immerse until
coverslip
separates

Image

Coverslipped
Slide

2.1 Imaging System Recommendations
The following table shows imaging systems used by 10x Genomics in the developmentof this protocol. Any equivalent imaging setup can be used as an alternative.
Supplier

Model

Configuration

Thermo Fisher Scientific

EVOS M7000

Inverted

Leica

Aperio Versa 8

Upright

Leica DMi8

Inverted

MetaSystems

MetaferUpright

Nikon

Nikon Eclipse Ti2Inverted

BioTekCytation 7

Inverted or Upright

Keyence

Keyence BZX800

Inverted

Fluorescence Recommended Configuration
Light source (or equivalent) with a wavelength range of 380–680 nm
Monochrome camera (14 bit, 2,424 x 2,424 pixel resolution)
DAPI filter cube (Excitation 392/23, Emission 447/60)
FITC filter cube (Excitation 480/40, Emission 535/50)
TRITC filter cube (Excitation 542/20, Emission 620/52)
Cy5 filter cube (Excitation 618/50, Emission 698/70)
2.18 �m/pixel minimum capture resolution
Exposure times 100 milli sec-2 sec
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Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

2.2 Imaging
a. Image all Capture Areas individually at the desired magnification using
fluorescence imaging settings. Ensure that fiducial frames are captured.
b. Consult the Visium Spatial Gene Expression for FFPE Imaging Guidelines
Technical Note (CG000436) for additional information.
c. After imaging, proceed immediately to the Coverslip Removal.

2.3 Coverslip Removala. Dispense 800 ml Milli-Q water in a beaker.
b. Remove the coverslip immediately after imaging is complete.
c. Immerse the slide sideways/horizontal in the beaker containing 800 ml Milli-Q
water with the coverslipped surface fully sideways.
d. Hold the slide in water until the coverslip slowly separates away from the slide.
To avoid damaging the tissue sections and Capture Areas or causing tissue
detachment, DO NOT move the slide up and down, shake forcibly or manually movethe coverslip.

ImmerseHold

Coverslip detaches

Coverslip
facing sideways

e. Gently immerse 15x in the Milli-Q water to ensure all mounting medium isremoved.
f.

Wipe the back of the slide with a laboratory wipe. Place on a flat, clean, nonabsorbent work surface and air dry.

g. Incubate slide on the Thermocycler Adaptor with the thermal cycler lid open for
3 min at 37°C.
h. Remove from the thermal cycler.
STOP

i.

Store the slide in a slide mailer with a desiccator or 50 -ml tube at 4°C for up to 2
weeks.

j.

Place the slide in the Visium Cassette. See Tips & Best Practices for assembly
instructions.

k. Add 100 µl 1X PBS along the side of the wells and proceed immediately to
Visium Spatial Gene Expression for FFPE User Guide (CG000407).
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CG000410 | Rev E

Troubleshooting
STEP

Notes

Tissue detachment• If tissue detachment is observed during the workflow, contact
support@10xgenomics.com.

2.2 Weak or no signal• Verify that samples were not exposed to light after staining withfluorescent antibodies.
• Verify antibody compatibility with decrosslinking conditions.
• Verify antibody dilutions. Ensure that antibody optimization is performed before immunofluorescence staining.
• Verify imaging system filter cubes and wavelength. Ensure thatfluorophores and filter cubes match.
• Protein of interest may have low expression. Test antibodies on
tissues of interest before working with Visium Spatial slides.

2.2 High background• Verify that samples did not dry out during the staining protocol.
Ensure that samples always remain covered in liquid.
• To prevent non-specific antibody binding, compare chosen antibody with antibodies that target the same cell type. If possible,
compare staining results to cells known to express higher or lower
levels of the target protein.

Results
Performing the Deparaffinization, Decrosslinking, Immunofluorescence Staining &Imaging protocol will likely result in a decrease in the number of unique transcripts
detected for many tissue types with no impact on the fraction of reads mapped
confidently to the transcriptome, as compared to the Deparaffinization, H&E
Staining, Imaging & Decrosslinking protocol (CG000409). However, this should notaffect interpretation of experimental results.
H&E

IF

H&E

IF

H&E

0.85 0.9

0.8

0.8

0.6
0.5
0.4
0.3
0.2

0.7
0.6
0.5
0.4

Relative UMI Counts

0.7

0.3
0.2

0.1

0.1

0.0

0.0

Mouse Brain

0.85

1.0
0.9
0.8
0.7
0.6

All Change of Median UMIs

0.9

0.9

0.85

0.77 0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

0.5 Breast
Human
Mouse
BrainCancer
0.4

H&E

1.0

Relative UMI Counts

0.9

IF

1.0

Median panel UMI count change
at 50k panel reads per spot

1.0

Relative UMI Counts

1.0

Relative UMI Counts

Relative UMI Counts

All UMIs
Changeof Median
UMI
All Change of Median
All Change
UMIsof Median
All Changeof Median
UMIs
All Changeof Median
UMIs

0.8

1.00

1.00

1.00

1.00

0.85

0.77

0.77

0.77

1.00

0.7
0.6
0.5

IF

0.85
0.77

0.4
0.3
0.2
0.1
0.0

Mouse Brain
Human Breast Cancer
Human Prostate Cancer
Human
Breast Human
Prostate
Mouse
Brain
Human
HumanBreast
Prostate
CancerCancerHuman Prostate Cancer
CancerCancer

Human
Human
Mouse
Breast
Prostate
BrainCancer
Cancer

0.3

H&E

IF

0.2 in UMI counts when downsampled to the same sequencing depth of 50,000 reads
Figure 1. All change
per spot.
0.1
0.0

Mouse Brain

Human Breast Cancer

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32

Demonstrated Protocol | Visium Spatial for FFPE – Deparaffinization, Decrosslinking, IF Staining & Imaging

CG000410 | Rev E

Document Revision Summary
Document Number

CG000410

Title

Visium Spatial Gene Expression for FFPE – Deparaffinization,
Decrosslinking, IF Staining & Imaging

Revision

Rev D to Rev E

Revision Date

September 2023

General Changes

Updated for general minor consistency of language and terms
throughout

Specific Changes• Updated Visium Spatial Gene Expression Slide Kit storage information
on page 2.

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