CG000312 • Rev F

DEMONSTRATED PROTOCOL

Methanol Fixation, Immunofluorescence Staining& Imaging for Visium Spatial Protocols
Overview

Visium Slide Selection

The Visium Spatial Gene Expression Solution measures thetotal mRNA in tissue sections and requires a Visium Spatialslide with intact tissue sections as input. Immunostaining
tissue sections with fluorescently labeled antibodies enables
simultaneous protein detection. This protocol outlines
methanol fixation, immunofluorescence staining, and imaging
of fresh frozen tissues for use with 10x Genomics Visium
Spatial protocols. Fixed and stained tissue sections are inputs
for the downstream Visium Spatial Tissue Optimization andVisium Spatial Gene Expression workflows.

Visium Spatial Tissue Optimization Slide (PN-3000394)

Additional Guidance• A readable label defines the active surface of the slide.
Tissue sections are always placed on the Capture Areason the active surface.
Etched
Frame
Label
on
CaptureActiveSurface
Areas

Consult the Visium Spatial Protocols - Tissue
Preparation Guide (Document CG000240) for Tips & Best
Practices on freezing, embedding, and cryosectioning
tissue and placing sections on Visium Spatial Slides.
Consult the Visium Spatial Gene Expression Imaging
Guidelines (Document CG000241) to verify imaging
settings before starting this Demonstrated Protocol.
Perform this Demonstrated Protocol on tissue sections
placed on the correct slide.
•

Use a plain glass slide if optimizing antibody
concentrations. Refer to the Antibody Optimization
section for more information.

•

Use a Visium Spatial Tissue Optimization Slide if
proceeding with tissue optimization.

•

Use a Visium Spatial Gene Expression Slide if
proceeding with library construction.

The
TissueOptimization
workflowmustbeperformed
beforethe
GeneExpression
workflow to determine the optimal tissue section
permeabilization
time.
Permeabilization
times
identified using the H&E staining protocol may not
be applicable to immunofluorescence staining.
After completing this protocol, proceed with either the
Visium Spatial Gene Expression Reagent Kits - Tissue
Optimization User Guide (CG000238) or the Visium Spatial• Used with Visium Spatial Gene Expression Reagent
Kits – Tissue Optimization User Guide (CG000238) to
identify optimal permeabilization time for a specifictissue type and section thickness.
• Includes 8 Capture Areas, each covered witholigonucleotides for mRNA capture.
• Each Capture Area is 8 x 8 mm and is surrounded byan etched frame.

Visium Spatial Gene Expression Slide (PN-2000233)• Used with Visium Spatial Gene Expression Reagent
Kits User Guide (CG000239) to generate Visium
Spatial Gene Expression libraries.
• Includes 2 or 4 Capture Areas, each with ~5,000
unique gene expression spots.
• Each Capture Area is 6.5 x 6.5 mm and is surrounded
by a fiducial frame for a total area of 8 x 8 mm.
• A readable label with a serial number defines theactive surface of the slide. Tissue sections are always
placed on the Capture Areas on the active surface.
Labelon
ActiveSurface

Gene Expression Reagent Kits User Guide (CG000239).
10xGenomics.com

Methanol Fixation, Immunofluorescence Staining & Imaging - Visium Spatial Protocols • Rev F

CaptureAreasCG000312 • Rev F

Visium Spatial Reagent Kits
Perform this Demonstrated Protocol in each step on tissue sections as listed.
Ensure that tissue sections have been placed onto the appropriate slide before
starting this Demonstrated Protocol. Consult the Visium Spatial Protocols - Tissue
Preparation Guide (CG000240) for more information.

Visium Spatial Tissue Optimization Slide Kit PN-1000191(store at ambient temperature)
VisiumSpatial Tissue Optimization Slide Kit

PNVisium Spatial Tissue Optimization
Slide

3000394*Slide Seals

3000279

Slide Cassette

3000406

Slide Gasket

3000426*Tissue Removal Buffer

2000221*Tissue Removal Enzyme

3000387*Not used in this protocol
10xGenomics.com

Visium Spatial Gene Expression Slide Kit, 16 rxns PN-1000185(store at ambient temperature or at –20°C according to kit label)
VisiumSpatial Gene Expression Slide Kit

PNVisium Spatial Gene Expression Slide

2000233*Visium Slide Seals, 40-pack or 20-pack

2000284/
3000279

Visium Slide Cassette & Gasket, 4-pack

2000282/
2000281(may come in varying configurations in different lots)*Not used in this protocol
10xGenomics.com

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Visium Spatial Reagent Kits
Perform this Demonstrated Protocol in each step on tissue sections as listed.

Visium Spatial Gene Expression Slide Kit, 4 rxns PN-1000188(store at ambient temperature or at –20°C according to kit label)
VisiumSpatial Gene Expression Slide Kit

PNVisium Spatial Gene Expression Slide

2000233*Visium Slide Seals, 12-pack or 5-pack

2000283
3000279

Visium Slide Cassette & Gasket, 1-pack

2000282/
2000281(may come in varying configurations in different lots)*Not used in this protocol
10xGenomics.com

10x Genomics
Accessories

Product

Part Number (Kit)

Thermocycler Adaptor
Visium Imaging Test Slide
Slide Alignment Tool

Part Number (Item)
3000380

1000194

2000235
3000433

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Workflow Overview

Tissue Preparation Guide

Imaging Guidelines

Section tissue onto slides.

Optimize imaging settings for
all Visium Spatial protocols.
Technical Note CG000241

Demonstrated Protocol CG000240

Methanol Fixation + ImmunofluorescenceFix and stain tissue. If IF staining for the first time,
optimize antibody concentrations.
Demonstrated Protocol CG000312
1st

2nd

Tissue Optimization

Library Construction

Identify optimal permeabilization time.

Construct Visium Spatial Gene Expression
libraries.
User Guide CG000239

User Guide CG000238

Visit the 10x Genomics Support website for the most updated documentation.

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Protocol Overview
Fixation

Perform this Demonstrated Protocol in each step on tissue sections as listed.

35 minIncubate 37°C
(1 min)Immerse &incubate -20°C(30 min)Place Slide in
Visium Slide
Cassette

Proceed to 1°
Antibody
Staining

MeOH chilled to -20°C

Primary Antibody Staining

45 minRepeat 4x

Add Wash Buffer(100 �l/well)

Add 1° Antibody Solution(50 �l/well) &incubate at room
temperature (30 min)Add blocking buffer(70 �l/well) &incubate at room
temperature (5 min)Removeblocking buffer

Secondary Antibody Staining

RemoveWash Buffer

Proceed to 2°
Antibody
Staining

Remove 1° Antibody
Solution

45 min(Skip if using fluorophore conjugated primary antibodies)
Repeat 4x

Add Wash Buffer(100 �l/well)

Add 2° Antibody Solution(50 �l/well) &incubate at room
temperature (30 min)Remove 2° Antibody
Solution

Coverslipping & Fluorescent Imaging

15 min*Add Mounting
Medium (200 �l)
and apply coverslip

Immerseslide 20x

RemoveWash Buffer

Immerse until
coverslip separates
Image

3X SSC Buffer

Proceed to
Coverslipping&
Remove Slide
Imagingfrom
Visium Slide
Cassette

Dip in 3X SSC one
additional time

Assemble in Visium
Slide Cassette
3X SSC Buffer

Add Wash Buffer(50 �l) to each well*Time excludes imaging steps

Proceed to 10x Genomics Visium Spatial Protocols
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Tips & Best Practices

Tips & Best Practices

Icons

TIPS

!

Tips & Best Practices
section includes
additional guidance

Signifies critical steprequiring accurate
execution

Troubleshooting section
includes additional
guidance

GeneralReagent
Handling• Thoroughly mix reagents before use.

PipetteCalibration• Follow manufacturer’s calibration and maintenance schedules.

Slide Storage• Store unused slides according to
instructions on the kit label, in original
packaging, and keep sealed. DO NOT
remove desiccant.

• Keep all enzymes, buffers and Master Mixes on ice during setup and use. Promptly
move reagents back to the recommended storage.

Store Unsealed Slide Mailers in a
Secondary Sealed Container• After tissue placement, store slides ina sealed container. If using an unsealed
slide mailer, store in a secondary sealedcontainer, such as a resealable bag.
• Store the sealed container containing
slides with tissue at −80°C for up to fourweeks.

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Tips & Best Practices

CG000312 • Rev F

Slide Handling• Always wear gloves when handling slides.

Active Surface with Tissue Sections• Exercise caution when handling slide
edges to prevent injury.
• 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.

Immersing Slide

Correct

Incorrect• Minimize exposure of the slides to sourcesof particles and fibers.

Not
immersed• When immersing slides in 3X SSC, ensurethat the tissue sections are completely
submerged.
• Keep the slide flat on a clean work surface
when adding reagents to the active surface.
• Ensure that no absorbent surface is incontact with the reagents on the slide
during incubation.

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Visium Slide
Cassette

Tips & Best Practices• The Visium Slide Cassette encases theslide and creates leakproof wells foradding reagents.
• Place the slides in the Visium Slide
Cassette only when specified.
• The Visium Slide Cassette may bereused once. Refer to the Visium Slide
Cassette and Visium Gasket Cleaning
section for instructions.
• An Insert Clip and four tabs at the backof the Visium Slide Cassette are used
for holding the slide in the Visium Slide
Cassette, as shown.

Visium Slide Cassette
Front

Back

Tab 3

Tab 1

InsertClip

VisiumGasket

Tab 4

Tab 2

Slide Alignment Tool

• The Visium Slide Cassette includes areusable Visium Gasket corresponding to
the Capture Areas on the slides.
• The Visium Slide Cassette may beassembled using the Slide Alignment
Tool or manually. Instructions for bothare provided in the following section.

Ridges• See Visium Slide Cassette Assembly &Removal instructions for details.
• Ensure that the back of the Visium
Slide Cassette is facing the user before
assembly. The active surface of the slidewith tissue sections will face down suchthat the slide label is no longer readable.
• Practice assembly with a 75 x 25 x 1 mm
plain glass slide.

Methanol Fixation, Immunofluorescence Staining & Imaging - Visium Spatial Protocols • Rev F

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Visium Slide
Cassette
Assembly

Tips & Best Practices

Position Visium Slide Cassettealong alignment tool ridges

1

Push Insert Clip along the ridge &press Visium Slide Cassette down

2

Press
down

!

Exercise caution
when handling slide edges
to prevent injury.

InsertClip

Ridge

InsertClip

Visium Slide Cassette secured onalignment tool3!

Slide insertion
may push Visium Gasket
out of alignment withslide cutouts. Adjust if
necessary.

Position Visium Gasket to align with
Visium Slide Cassette cutouts

4

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

Press slide down until it is flushwith the Visium Gasket and undertabs 3 & 4

5

6

Slide
label

Tab 1
Tab 2

Press down

Tab 3
Tab 4

Active surface
faces Visium
GasketRemove Visium Slide Cassette
while pressing slide against the
Visium Gasket

7

Pull up

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Tips & Best Practices

CG000312 • Rev F

Visium Slide
Cassette
RemovalPosition Visium Slide Cassettealong alignment tool ridges

12

Insert
Clip

Press
down

Ridge

Visium Slide Cassette sits securely
on alignment tool

3

Push Insert Clip along the ridge& press down

Lift slide at Visium Slide Cassettegroove4

GrooveMethanol Fixation, Immunofluorescence Staining & Imaging - Visium Spatial Protocols • Rev F

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CG000312 • Rev F

ManualVisium Slide
Cassette
Assembly &RemovalTips & Best Practices

Assembly

Visium Slide Cassette
Front

i. Remove the Visium Gasket from the
Visium Slide Cassette and re-insert
the Visium Gasket, ensuring that
the Visium Gasket and Visium Slide
Cassette cutouts are aligned.
ii. Align the label on top of the slide to
the top of the Visium Slide Cassette,
as shown.
iii. Insert the slide under tabs 1 and 2.
Ensure that the long edge of the slide
is flush with the side of the Visium
Slide Cassette.
iv. Press the insert clip very firmly byapplying even force on the lower partof the insert clip.

i. Press the insert clip very firmly torelease the slide from the Visium
Slide Cassette.

Tab 3

Tab 1

InsertClip

VisiumGasket
Tab 2

Tab 4

Visium Slide Cassette Assembly
Squeeze
InsertClip

v. Place a finger in between tab 3 andthe top of the Visium Slide Cassette,
and one finger between tab 4 and thebottom of the Visium Slide Cassette.
Press down on the slide evenly
until the slide is under each tab andrelease the insert clip.
RemovalBack

Label facing
down

Slide
under tabs
Insert Clip - Press Firmly
Lift
here
Press
firmlyii. Lift slide at Visium Slide Cassettegroove between tabs 3 and 4 until theslide can be removed.

Methanol Fixation, Immunofluorescence Staining & Imaging - Visium Spatial Protocols • Rev F

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Tips & Best Practices

CG000312 • Rev F

Visium Slide
Cassette &Visium Gasket
Cleaning

Remove slide from Visium
Slide Cassette1Mark the top portion of the Visium
Gasket that faced the slide with an
alcohol resistant marker

2

Remove Visium Gasket

33

44

Rinse Visium Slide Cassetteand Visium Gasket withultrapure water

GrooveSpray with 70% isopropanol, thenrinse with ultrapure water

45Groove37

Spray with 70% isopropanol a second
time, then rinse with ultrapure water46

GrooveAir dry

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

8

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Tips & Best Practices

Reagent
Addition &Removal fromWells

• Place the assembled slide in the Visium
Slide Cassette flat on a clean work
surface.
• Dispense and remove reagents along
the side of the wells without touching thetissue sections and without introducing
bubbles.

Reagent Addition/Removal
Add/remove
along the sideof wells• Always cover the tissue section
completely when adding reagents to thewell. A gentle tap may help spread thereagent more evenly.

!

• Pipette reagent carefully so that tissue is
not disrupted or dislodged.

DO NOT
introduce
air bubbles• Ensure that no bubbles are introduced in
the process.

Slide Incubation
Guidance

Incubation at a specified temperature!

•

Position a Thermocycler Adaptor on athermal cycler that is set at the incubation
temperature.

•

Ensure that the Thermocycler Adaptor isin contact with the thermal cycler surface
uniformly.

•

When incubating a slide, position the slideon the Thermocycler Adaptor with theactive surface facing up.

•

Ensure that the entire bottom surface ofthe slide is in contact with Thermocycler
Adaptor.

•

When incubating a slide encased in a
Visium Slide Cassette, place the assembled
unit on the Thermocycler Adaptor with thewells facing up. The Visium Slide Cassetteshould always be sealed when on the
Thermocycler Adaptor.

Place Thermocycler Adaptor

Incubate Slide

Incubate Assembled Visium Slide
Cassette

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Antibody Optimization
Optimization of antibody concentration is critical for executing this Demonstrated Protocol.
•

Optimal antibody concentrations for this Demonstrated Protocol may differ from otherapplications due to shortened antibody incubation periods. Composition of reagents andfixatives may differ from other immunofluorescence applications.

•To optimize antibody concentration, draw representative frames on the back of 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 Slide Cassette. Ensure that tissue sections used during optimization are similar insize to tissue sections used for Tissue Optimization and Gene Expression Protocols•

Primary antibody: Execute the Tissue Fixation & Immunofluorescence Staining
protocol (steps 1.0-1.5) using a range of primary antibody concentrations. 10x Genomics
recommends performing an antibody dilution series with a starting concentration of 0.01
�g/�l (0.5 �g/sample). 0.01 �g/�l corresponds to the 1:50 dilution in the image below.

•

Secondary antibody: Use a similar range of concentrations for secondary antibodies orrefer to the manufacturer's recommended dilution.

•

Select the antibody concentration that results in the specific staining of desired cells,
while minimizing nonspecific background staining.

•

Wash Visium Slide Cassette and Visium Gasket after antibody staining. See Tips & Best
Practices for more information.

•

An example dilution layout is provided below. DAPI image is provided to show presenceof tissue for each antibody dilution. Dilutions are of recombinant Anti-NeuN antibody
conjugated to Alexa Fluor 488 (Abcam, PN:190195, 0.5 mg/ml). A 1:100 dilution (0.25 �g/sample) was considered optimal in this example.

Example fluorophore conjugated primary
antibody dilution series.
1:2000

1:1000

1:500

1:200

1:100

1:75

1:50

No AB

28.5 mm

ExampleSlide Layout

8 mm

8 mm

11.25 mm

1.3 mm

3.5 mm

1.5 mmMethanol Fixation, Immunofluorescence Staining & Imaging - Visium Spatial Protocols • Rev F

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Tissue Fixation & Immunofluorescence Staining

Tissue Fixation & Immunofluorescence Staining
1.0 Overview
1.1 Specific Reagents & Consumables
1.2 Tissue Fixation
1.3 Immunofluorescence Staining - Primary and Secondary Antibodies
1.4 Immunofluorescence Staining - Fluorophore Conjugated Primary Antibodies
1.5 Slide Mounting & Coverslip Application
Ensure that this protocol is performed on tissue sections on the correct slide. Referto the Introduction, Workflow Overview, and Antibody Optimization sections for more
information.

1.0 Overview

Plain Glass Slide

Visium Spatial Tissue
Optimization Slide

Visium Spatial Gene
Expression Slide

Methanol Fixation,
Immunofluorescence Staining &Imaging Protocol
Ensure that microscope settings have been verified and imaging programs
have been created before starting this protocol. Consult the Visium Spatial Gene
Expression Imaging Guidelines Technical Note (CG000241) for more information.

!

If proceeding to Tissue Optimization, DO NOT add buffers or staining reagents towell A1, which is reserved for the positive RNA control. Addition of these reagents tothis well may prevent visualization of the positive control.
If staining using fluorophore conjugated primary antibodies, proceed directly to step
1.4 after completing step 1.2.

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1.1
Specific Reagents& Consumables

Tissue Fixation & Immunofluorescence Staining

The items in the table below have been validated by 10x Genomics and are highly
recommended for Visium Spatial Reagent Kits protocols. Substituting materials may
adversely affect system performance. This list does not include standard laboratory
equipment, such as water baths, centrifuges, pH meters, vortex mixers, freezers, etc.
Ensure that tissue sections have been placed on the appropriate slide and stored at -80°C.
The slide will be retrieved in step 1.2.
Tissue Fixation
VendorItem

Millipore Sigma Methanol, for HPLC, ≥99.9%

Part Number
34860

Tissue IF Staining
Vendor

Item

Part Number

Corning

Self-Standing Polypropylene Centrifuge Tubes, 50 ml, sterile

430921Sigma Aldrich

Triton X-100 Solution, ~10% in H2O

93443-100ML

Thermo Fisher
Scientific

Thermo Scientific Signature Series Cover Glasses
Shandon ColorFrost Plus Slides(Alternatively, use any 75 x 25 x 1 mm slide)
Fisherbrand Superfrost Plus Microscope Slides(Optional - Alternatively, use any 75 x 25 x 1 mm slide)
DAPI Solution (1 mg/ml)
RiboLock RNase Inhibitor(Alternative to Millipore Sigma product)
Tween 20 Surfact-Amps Detergent Solution(Alternative to Sigma Aldrich Triton X-100)

22-050-233
677621412-550-15
62248
EO038228320

Millipore Sigma SSC Buffer 20x Concentration
Protector RNase Inhibitor
Albumin, Bovine Serum, 10% Aqueous Solution, Nuclease-Free(Alternative to Miltenyi product)

S6639
3335402001
126615-25ML

Miltenyi Biotec

MACS BSA Stock Solution

130-091-376

BioLegend

Human TruStain FcX (Fc Receptor Blocking Solution)(Alternatively, use any species appropriate Fc blocking solution)
TruStain FcX (anti-mouse CD16/32) Antibody (discontinued)
TruStain FcX PLUS (anti-mouse CD16/32) Antibody

422301

New England
Biolabs

Ribonucleoside Vanadyl Complex
RNase Inhibitor, Murine(Alternative to Millipore Sigma product)

S1402S
M0314L-Primary Antibodies--

Secondary Antibodies-

101309156603

Additional Materials-

Dry Ice--

Glycerol--

Isopropanol--

Ultrapure/Milli-Q water (from Milli-Q Integral Ultrapure Water
System or equivalent)--

Forceps-

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Recommended
Thermal Cyclers

Tissue Fixation & Immunofluorescence Staining

Supplier

Description

Part Number

Bio-RadC1000 Touch Thermal Cycler
with 96-Deep Well Reaction
Module1851197

Analytik Jena†

Biometra TAdvanced 96 SG

846-x-070-241(x=2 for 230 V; 4 for 115 V; 5 for 100 V, 50-60 Hz)

Eppendorf‡

Thermo Fisher
Scientific

Mastercycler X50s

1000518

MasterCycler Pro(discontinued)

North America 950030010
International 6321 000.019

Veriti 96-Well Thermal Cycler(discontinued)

4375786

For select instruments, ramp rates should be adjusted for all steps as described below:†Analytik Jena Biometra TAdvanced 96 SG: 2°C/sec for both heating and cooling‡Eppendorf Mastercycler X50s: 3°C/sec heating and 2°C/sec coolingPreparation - Buffers

PrepareMethanol• Dispense 40 ml/slide in a 50-ml centrifuge tube. Chill to -20°C before use.

Ribonucleoside
VanadylComplex• Incubate sealed vial at 65°C for 10
min or until solution is reconstituted
to a dark green solution with novisible particulates. DO NOT exceed
15 min. Aliquot into single-use tubes(550 �l per slide including secondary
staining). Store tubes at -20°C. DO
NOT exceed 2 freeze-thaw cycles peraliquot. If using an aliquot immediately
for step 1.3, no freezing is necessary.
Store at room temperature until use. If
precipitation is observed, incubate at
65°C for 5 min.

2X Blocking Buffer(prepare fresh on ice)
2X Blocking
Buffer(prepare on ice)

Stock

BeforeIncubation

Final

Nuclease-free Water

After
Incubation

2X + 20%(μl)

4X + 20%(μl)

8X + 20%(μl)

336.7 �l673.4 �l

1,346.9 �l1,756.8 �l

SSC Buffer

20X

6X

439.2 �l878.4 �l

BSA

10%

4%

585.6 �l

1,171.2 �l 2,342.4 �l

Triton X-100
or Tween 20

10%

0.2%

29.3 �l58.6 �l117.7 �l

RNase Inhibitor

40 U/�l 2 U/�l

73.2 �l146.4 �l292.8 �l

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Tissue Fixation & Immunofluorescence Staining

Preparation - Buffers
3X SSC

Mounting
Medium3X SSC

Stock

Final

550 ml(1 slide)

SSC Buffer

20X

3X

82.5 ml

Ultrapure Water--

467.5 ml

Mounting Medium

Stock

Final

200 �l(1 slide)

Glycerol

100%

85%

170 �lRNase Inhibitor

40 U/�l

2 U/�l10 �lNuclease-free Water

20 �l

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1.2
Tissue Fixation

Tissue Fixation & Immunofluorescence Staining!

If using a Visium Spatial Gene Expression
Slide, note the serial number on the slidelabel; will be required for downstream
analysis.

Place Thermocycler Adaptor

Ensure that the methanol (40 ml/slide)dispensed in a 50-ml centrifuge tube ischilled to -20°C.
a. Place a Thermocycler Adaptor on athermal cycler set at 37°C and equilibrate
for 5 min. Heating the thermal cycler lid is
not required.

Incubate Slide for 1 min at 37°C
DO NOT
close lidb. Remove slide from -80°C and place on dry
ice in a sealed container.

!

Delay in transferring slides to dry ice may
result in condensation, which may cause
tissue damage and/or shifting of tissue
sections on the slide.
c. Place slide on the Thermocycler Adaptor
with the active surface facing up andincubate 1 min at 37°C. DO NOT close thethermal cycler lid. Maintain thermal cycler
at 37°C for step 1.2.

Incubate in Methanol for 30 min at −20°C

d. Remove slide from Thermocycler Adaptor
and if necessary, wipe excess liquid fromthe back of the slide, without touching thetissue sections.
e. Completely immerse the slide in the prechilled methanol. Secure the tube cap toprevent methanol loss.
f. Incubate 30 min at -20°C.

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TIPS
1.3
Immunofluorescence
Staining - Primary andSecondary Antibodies!

If using
fluorophore conjugatedprimary antibodies,
proceed directly to step 1.4!

RIbonucleoside
Vanadyl Complex stock
solution will precipitate
if left at ambient or cooltemperatures for extended
periods of time. Oncethe solution has cooled,prepare 1X Blocking
Buffer and Wash Buffer
immediately.

!

Ensure that tissue
sections do not dry out
throughout the staining
protocol

Tissue Fixation & Immunofluorescence Staining

a. Place the slide in the Visium Slide Cassette. See Tips & Best Practices for assembly
instructions. Practice assembly with a blank slide.
b. Remove Ribonucleoside Vanadyl Complex aliquots from -20°C and place at 65°C
for 5 min until the solution is reconstituted to a dark green solution with no visible
particulates. DO NOT exceed 10 min. Allow the solution to cool to ambient temperature
before use and proceed immediately to the next step.
c. Prepare 1X Blocking Buffer on ice.
1X Blocking Buffer
Vortex.

1X(μl)

2X + 15%(μl)

4X + 15%(μl)

8X + 15%(μl)

2X Blocking Buffer

35.0

80.5

161.0

322.0

Nuclease-free Water

26.0

59.8

119.6

239.2

Human or Mouse TruStain FcX*

2.0

4.6

9.2

18.4

Ribonucleoside Vanadyl Complex

7.0

16.1

32.2

64.4

Total

70.0

161.1

322.0

644.0

*Choose FC blocking reagent based on sample species.
d. Prepare Wash Buffer on ice.
Wash Buffer
Vortex.

1X(μl)

2X + 15%(μl)

4X + 15%(μl)

8X + 15%(μl)

2X Blocking Buffer

525.0

1,207.5

2,415.0

4,830

Nuclease-free Water

420.0

966.0

1,932.0

3,864

Ribonucleoside Vanadyl Complex

105.0

241.5

483.0

966

Total

1,050.0

2,415.0

4,830.0

9,660

e. Vortex 1X Blocking Buffer and add 70 µl along the side of the wells to uniformly cover
the tissue sections, without introducing bubbles. Tap gently to ensure even coverage.
f. Incubate for 5 min at room temperature.
g. Prepare Primary Antibody Solution on ice. Pipette mix.
Primary Antibody Solution.
Vortex, centrifuge briefly.

1X
(μl)

2X + 10%(μl)

4X + 10%(μl)

8X + 10%(μl)

2X Blocking Buffer

25.00

55.00

110.00220.00

Nuclease-free Water

Variable

Variable

Variable

Variable

Primary Antibody #1

Variable

Variable

Variable

Variable

Primary Antibody #2 (optional)

Variable

Variable

Variable

Variable

Primary Antibody #3 (optional)

Variable

Variable

Variable

Variable

RNase Inhibitor

6.75

14.85

29.70

59.40

Total

50.00

110.00220.00440.00

Antibody volumes will depend on concentrations determined during antibody
optimization. Add an appropriate volume of nuclease-free water based on theamount of added antibody to achieve the stated total volume.
h. Remove 1X Blocking Buffer.
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Tissue Fixation & Immunofluorescence Stainingi. Add 50 µl Primary Antibody Solution along the side of each well. Tap gently to ensure
uniform coverage.
j. Incubate for 30 min at room temperature.
k. Prepare Secondary Antibody Solution on ice. Pipette mix. Avoid light exposure.
Secondary Antibody Solution.
Vortex, centrifuge briefly.

1X
(μl)

2X + 10%(μl)

4X + 10%(μl)

8X + 10%(μl)

2X Blocking Buffer

25.00

55.00

110.00220.00

Nuclease-free Water

Variable

Variable

Variable

Variable

Secondary Antibody #1

Variable

Variable

Variable

Variable

Secondary Antibody #2 (optional)

Variable

Variable

Variable

Variable

Secondary Antibody #3 (optional)

Variable

Variable

Variable

Variable

DAPI

0.17

0.38

0.75

1.50

RNase Inhibitor

6.75

14.85

29.70

59.40

Total

50.00

110.00220.00440.00

Antibody volumes will depend on concentrations determined during antibody
optimization. Add an appropriate volume of nuclease-free water based on the amount ofadded antibody to achieve the stated final volume.
l. Remove the Primary Antibody Solution.

!

m. Add 100 µl Wash Buffer along the side of each well.

Ribonucleoside
Vanadyl Complex settles
rapidly in solution. Briefly
vortex Wash Buffer before
each wash.

n. Remove Wash Buffer.
o. Repeat steps m and n four more times for a total of five washes.
p. Add 50 µl Secondary Antibody Solution along the side of each well. Tap gently toensure uniform coverage.
q. Incubate for 30 min at room temperature. Avoid light exposure.
r. Remove the Secondary Antibody Solution.
s. Add 100 µl Wash Buffer along the side of each well.
t. Remove Wash Buffer.
u. Repeat steps s and t four more times for a total of five washes. Save 50 µl Wash
Buffer per sample for step 2.2.
v. Proceed immediately to step 1.5

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1.4
TIPS
Immunofluorescence
Staining - Fluorophore
Conjugated Primary
Antibodies!

RIbonucleoside
Vanadyl Complex stock
solution will precipitate
if left at ambient or cooltemperatures for extended
periods of time. Oncethe solution has cooled,prepare 1X Blocking
Buffer and Wash Buffer
immediately.

Tissue Fixation & Immunofluorescence Staining

a. Place the slide in the Visium Slide Cassette. See Tips & Best Practices for assembly
instructions. Practice assembly with a blank slide.
b. Remove Ribonucleoside Vanadyl Complex aliquots from -20°C and place at 65°C
for 5 min until the solution is reconstituted to a dark green solution with no visible
particulates. DO NOT exceed 10 min. Allow the solution to cool to ambient temperature
before use and proceed immediately to the next step.
c. Prepare 1X Blocking Buffer on ice.
1X Blocking Buffer
Vortex.

1X(μl)

2X + 15%(μl)

4X + 15%(μl)

8X + 15%(μl)

2X Blocking Buffer

35.0

80.5

161.0

322.0

Nuclease-free Water

26.0

59.8

119.6

239.2

Human or Mouse TruStain FcX*

2.0

4.6

9.2

18.4

Ribonucleoside Vanadyl Complex

7.0

16.1

32.2

64.4

Total

70.0

161.1

322.0

644.0

*Choose FC blocking reagent based on sample species.
d. Prepare Wash Buffer on ice.

!

Ensure that tissue
sections do not dry out
throughout the staining
protocolWash Buffer
Vortex.

1X(μl)

2X + 15%(μl)

4X + 15%(μl)

8X + 15%(μl)

2X Blocking Buffer

275

632.5

1,265

2,530

Nuclease-free Water

220

506.0

1,012

2,024

Ribonucleoside Vanadyl Complex

55

126.5

253

506

Total

550

1,265.0

2,530

5,060

e. Vortex 1X Blocking Buffer and add 70 µl along the side of the wells to uniformly cover
the tissue sections, without introducing bubbles. Tap gently to ensure even coverage.
f. Incubate for 5 min at room temperature.
g. Prepare Primary Antibody Solution on ice. Pipette mix. Avoid light exposure.
Primary Antibody Solution.
Vortex, centrifuge briefly.

1X
(μl)

2X + 10%(μl)

4X + 10%(μl)

8X + 10%(μl)

2X Blocking Buffer

25.00

55.00

110.00220.00

Nuclease-free Water

Variable

Variable

Variable

Variable

Primary Antibody #1

Variable

Variable

Variable

Variable

Primary Antibody #2 (optional)

Variable

Variable

Variable

Variable

Primary Antibody #3 (optional)

Variable

Variable

Variable

Variable

DAPI

0.17

0.38

0.75

1.50

RNase Inhibitor

6.75

14.85

29.70

59.40

Total

50.00

110.00220.00440.00

Antibody volumes will depend on concentrations determined during antibody
optimization. Add an appropriate volume of nuclease-free water based on theamount of added antibody to achieve the stated final volume.
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CG000312 • Rev F

Tissue Fixation & Immunofluorescence Staining

h. Remove 1X Blocking Buffer.

!

i. Add 50 µl Primary Antibody Solution along the side of each well. Tap gently to ensure
even coverage.

Ribonucleoside
Vanadyl Complex settles
rapidly in solution. Briefly
vortex Wash Buffer before
each wash.

j. Incubate for 30 min at room temperature. Avoid light exposure.
k. Remove the Primary Antibody Solution.
l. Add 100 µl Wash Buffer along the side of each well.
m. Remove Wash Buffer.
n. Repeat steps l and m four more times for a total of five washes. Save 50 µl Wash
Buffer per sample for step 2.2.
o. Proceed immediately to step 1.5

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Tissue Fixation & Immunofluorescence Staining

1.5
Slide Mounting &Coverslip Application

a. Dispense the following volumes of 3X SSC
Buffer.
50 ml in one 50-ml centrifuge tube/slide
500 ml in a beaker/2 slides.

Cover uniformly with Mounting
Mediumb. Remove the slide from the Visium Slide
Cassette. See Tips & Best Practices for more
information.
c. Immerse the slide 20x in the 3X SSC Buffer inthe 50-ml centrifuge tube.

Apply coverslip

d. Wipe excess liquid from the back of the slide
without touching the tissue section. Some
droplets may remain.
e. Add 200 µl Mounting Medium to cover thetissue sections on the slide uniformly. If
necessary, hold the slide at an angle foruniform coverage.

Press downf. Apply the coverslip at an angle on one end of
the slide. Slowly lower the coverslip, pressing
down gently with forceps, without introducing
bubbles.
g. Remove excess Mounting Medium by placing
Remove excess Mounting Medium
one long edge of the slide on a laboratory
wipe, and gently tilt the slide back and forth.
Repeat with the second long edge of theslide. Repeat the process until the coverslip issecured.
h. After the coverslip is secured, immediately
proceed with imaging.

!

DO NOT let the attached coverslip dry.
DO NOT use Cytoseal, nail polish, or any otherpermanent mounting medium for securing
the coverslip.

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2. Tissue Imaging
2.0 Imaging System Recommendations
2.1 Tissue Imaging
2.2 Coverslip Removal
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. Imaging
systems should have both brightfield and fluorescence capacity. Required
fluorescence channels will depend on antibody selection. Exposure times will dependon antibody sensitivity and fluorophores.

2.0 Imaging System
Recommendations

Supplier

Description

Nikon

Nikon Eclipse Ti2

Molecular Devices

ImageXpress Nano Automated Cell Imaging System

Hamamatsu

NanoZoomer S60

Keyence

Keyence BZX800

BioTekCytation 7

Thermo Fisher Scientific

EVOS M7000

Leica

Leica DMi8
Versa 8

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/GFP filter cube (Excitation 466/40, Emission 698/70) (Optional - dependent on antibody selection)
TRITC filter cube (Excitation 542/20, Emission 620/52)
Cy5 filter cube (Excitation 618/50, Emission 698/70) (Optional - dependent on antibody selection)
2.18 �m/pixel minimum capture resolution
Exposure times 100 milli sec-2 sec• If imaging a Visium Spatial Tissue Optimization Slide or blank slide for antibody
optimization, image all Capture Areas together at the desired magnification using
fluorescence imaging settings.

2.1
Tissue Imaging• If imaging a Visium Spatial Gene Expression Slide, image all Capture Areas
individually at the desired magnification using fluorescence imaging settings. Ensurethat fiducial frames are captured.
• Images should be saved as a multi-channel image (multi-image TIFF), merged RGB
image, or as individual single channel images (grayscale TIFF).
• Consult the Visium Spatial Gene Expression Imaging Guidelines Technical Note
(CG000241) for additional information.

!

• Proceed immediately to next step if continuing with the Visium Spatial Tissue
Optimization or Gene Expression workflows.

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CG000312 • Rev F

Tissue Imaging

a. Wash Visium Slide Cassette and Visium
Gasket. See Tips & Best Practices for more
information.

2.2
Coverslip Removalb. Immerse the slide sideways in the beakercontaining 3X SSC with the coverslippedsurface fully sideways.
c. Hold the slide in 3X SSC Buffer until thecoverslip slowly separates away from theslide.
To avoid damaging the tissue sections andCapture Areas or causing tissue detachment, DO
NOT move the slide up and down, shake forciblyor manually move the coverslip.

Immerse in 3X SSC Buffer

Coverslip
facingsideways

Hold in 3X SSC Buffer

d. Remove the slide from the 3X SSC Buffer andimmerse 1x in the 3X SSC Buffer to ensure all
Mounting Medium is removed.
e. Wipe excess liquid from the back of the slide,without touching the tissue sections.

Coverslip detaches

f. Place slide in a clean Visium Slide Cassette.
g. Add 50 µl Wash Buffer along the side of thewells.
DO NOT add Wash Buffer to well reserved for
positive RNA control if proceeding to Tissue
Optimization.

!

h. Proceed immediately to the Visium Spatial
Tissue Optimization User Guide (CG000238)
or the Visium Spatial Gene Expression User
Guide (CG000239).

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CG000312 • Rev F

Results

Results
Performing the Methanol Fixation, Immunofluorescence Staining & Imaging protocol
will likely result in a decrease in the number of unique transcripts detected for manytissue types, as compared to the Methanol Fixation, H&E Staining & Imaging protocol(CG000160). However, this should not affect interpretation of experimental results.
All % Reads Mapped Confidently toTranscriptome

Median UMIs per Spot

% Reads Mapped Confidently
to Transcriptome

All Median UMIs

Mouse
Brain

Human
Brain

Human
Breast Cancer

Mouse
Brain

Human
Brain

Human
Breast Cancer

Figure 1. Median UMI counts and % reads mapped confidently to transcriptome for mousebrain, human brain, and human breast cancer tissue. Values indicate the average of 4
replicates per condition. UMI metrics are based on samples downsampled to 20K raw
reads per spot.

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CG000312 • Rev F

Troubleshooting

Troubleshooting
STEP

NOTES

2.1
Weak or no signal• Verify that samples were not exposed to light after staining with fluorescentantibodies.
• Verify antibody dilutions. Ensure that antibody optimization is performed before
immunofluorescence staining.
• Verify that the correct fluorescent secondary antibody was used at the correct
dilution, if applicable.
• Verify imaging system filter cubes and wavelength. Ensure that fluorophores andfilter cubes match.
• Protein of interest may have low expression. Test antibodies on tissues of interest
before working with Visium Spatial slides.

2.1
High background• Verify that samples did not dry out during the staining protocol. Ensure thatsamples 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.

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Contact:
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The use of 10x Genomics products in practicing the methods set forth herein has not been validated by 10x Genomics, and such non-validated use is NOT COVEREDBY 10X GENOMICS STANDARD WARRANTY, AND 10X GENOMICS HEREBY DISCLAIMS ANY AND ALL WARRANTIES FOR SUCH USE. Nothing in this document should beconstrued as altering, waiving or amending in any manner 10x Genomics terms and conditions of sale for the Chromium Controller or the Chromium Single Cell Controller, consumables or software, including without limitation such terms and conditions relating to certain use restrictions, limited license, warranty and limitation of
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