CG000663 | Rev A

DEMONSTRATED PROTOCOL

Visium CytAssist Spatial Gene Expression for
Fixed Frozen – Tissue Preparation Guide
Introduction
The Visium CytAssist Spatial Gene Expression for Fixed Frozen assay is designed to analyze mRNA intissue sections derived from fixed frozen tissue samples. Proper tissue handling and preparation techniquespreserve the morphological quality of the tissue sections and the integrity of mRNA transcripts. This is
critical for downstream library preparation and generation of high quality sequencing data using the
Visium CytAssist Spatial Gene Expression protocols.
The Tissue Preparation Guide provides guidance on:• Best practices for handling tissue samples and slides before and after cryosectioning.
• Tested blank slides compatible with the Visium CytAssist Spatial Gene Expression assay.
• Fixing, cryopreserving, and embedding of tissue samples prior to cryosectioning.
• Cryosectioning of tissue samples and placement of sections on blank slides.
• Assessment of tissue block RNA quality.

Additional GuidanceContents

This protocol was demonstrated with several tissue
types (visit the 10x Genomics support website for a
detailed list). Additional optimization may berequired for the preparation of tissues not tested.

1
2

Introduction
Tips & Best Practices

45

Determining Allowable Area
Visium CytAssist Tested Slides

6

1. Tissue Fixing & Embedding

The slides prepared using the Tissue Preparation
Guide can be used with:

7
8
11

1.1 Reagents & Consumables
1.2 Tissue Fixation
1.3 Tissue Freezing & Embedding• Visium CytAssist Spatial Gene Expression for
Fixed Frozen – Rehydration, H&E Staining,
Imaging & Decrosslinking Demonstrated Protocol(CG000662)

13

2. Cryosectioning & Section Placement

14
15
17
19
19
20

2.1 Reagents & Consumables
2.2 Cryosectioning
2.3 Section Placement
Leftover Tissue Block Storage
Shipping Guidelines
2.4 RNA Quality Assessment

23

Troubleshooting• Visium CytAssist Spatial Gene Expression User
Guide (CG000495), Rev D or later

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Tips & Best Practices

Tips & Best Practices
section includes additional
guidance

Signifies critical steprequiring accurate
execution

Troubleshooting section
includes additional
guidance

Best Practices

Section Placement on Blank Slides• Best practices for handling any tissues include
using sterile techniques, nuclease-free reagents,
and consumables.

• After section placement, blank slides are referred
to as tissue slides.

Cryosectioning Temperature• Cryosectioning temperatures impact tissue section
integrity. A temperature setting of –10°C to –20°C
for blade and –10°C to –20°C for the specimen headis recommended.
• The temperature settings depend upon thelocal conditions, tissue types, and the cryostat
used. Settings should be optimized based on thequality of resulting tissue sections.
• During prolonged sectioning periods, allow thecryostat temperature to equilibrate by briefly
closing the chamber.

Sectioning Speed• Sectioning speed depends upon the desired
thickness of the sections and the condition of thetissue. Thicker sections require a slow sectioning
speed.
• Faster sectioning speed may lead to cracks or tears inthe sections or damage to the tissue block or cryostat.

Section Thickness• Recommended section thickness for most tissue
types is 10 µm, but tissues from 10−20 µm arecompatible with the assay. Sections outside ofthat range may result in reduced performance.

• Prior to section placement, draw an outlineof the allowable area on the back of the blankslide to ensure downstream compatibility with
the Visium CytAssist Tissue Slide Cassetteand Visium CytAssist instrument (refer to
Determining Allowable Area). Drawing should be
removed after tissue placement.
• If working with sections on multiple tissue slides,
ensure that sections are placed in the same
location on the tissue slides for improved imaging
efficiency.
• Only one section from each tissue slide can beused for each Visium CytAssist Spatial Gene
Expression Slide Capture Area.

Practice Section Placement• Practice correct section placement using nonexperimental blocks.
• Practicing section placement also allows fordetermining an ideal tissue thickness for thetissue type or block.
• Sections should be placed on the compatible
blank slides listed in the Specific Reagents andConsumables section or similar slides within
specified dimensions. For more information, refer
to Visium CytAssist Tested Slides.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Handling Tissue Slides• Keep tissue slides on dry ice.
• If placing in a slide mailer or 50-ml centrifuge
tube after sectioning, mailer or tube should be
pre-cooled to cryostat temperature for 10–15 min.
• Immediately place storage container on dry
ice for transport to a -80°C freezer for longterm storage.
• Avoid tissue slides touching one anotherwhile in storage.

RNA Quality Assessment• Assess RNA quality of the tissue block before
placement of sections on blank slides by
calculating the percentage of total RNA
fragments >200 nucleotides (DV200) of RNA
extracted from tissue sections.
• Various factors could lead to variations in DV200
scores, such as specific tissue types, diseased or
necrotic tissues, excess OCT, sample preparation,
handling, loading concentration errors, or errors
with ladder.

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3Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Determining Allowable Area
Use the following diagrams to verify that freshly placed tissue sections are compatible. Reference the images
below to draw the allowable area on the back of blank slides (remove after tissue placement). Images are to scale if
scaling settings are not modified. To verify, ensure that the first block in section A measures 6.5 x 6.5 mm.

A Ensure that the area of interest will fit within the Capture Area of a Visium CytAssist Spatial Gene Expression Slide.
Tissue sections larger than these Capture Areas may be placed on the slide, but only the tissue within the Capture Area will be processed by
the CytAssist instrument.
Overlay the slide, centering the tissue on either square.
6.5 mm11 mm
Capture Area
Capture Area

B If slide has no frosted areas,

If slide has a frosted end and/or marks,
overlay on diagram 1 and then on diagram 2.
Check the allowable area from both the top (1) and bottom (2) toensure the tissue lies within the green allowable area (area is variable
due to variability in the dimensions of frosted areas across slide brands):
15 mm from edge of frosted area/marks
5 mm from the sides

5 mm
15 mm

5 mm

15 mm

5 mm

( Diagram 2 )align bottom edgeof frosted area* to thehorizontal black line

5 mm

Tissueoutsidethis region
cannot beanalyzed( Diagram 1 )

5 mm

5 mm

15 mm

overlay on this diagram.
Tissue should lie within the green allowable area:
15 mm from top and bottom edges
5 mm from the sides

+

+

15 mm

align bottom edge** tothe horizontal black line
*If text is present below frosted
area of slide, align bottom of thetext to the line

C Examples of Good Tissue Placement

Examples of Bad Tissue Placement

Unfrosted Slide

Unfrosted Slide

Frosted Slide**If markings are present atbottom edge of slide, alignmarkings to the + signs

Frosted Slide
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4Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Visium CytAssist Tested Slides
The following slides have been tested for use with the Visium CytAssist Tissue Slide
Cassette and instrument.
Item

Length (mm)Width (mm)Thickness (mm)Epredia Shandon ColorFrost Plus

75.0

25.0

1.0

Fisherbrand SuperFrost Plus

75.0

25.0

1.0

Sigma-Aldrich Poly Prep Slides

75.0

25.0

1.0

VWR SuperFrost Plus Micro Slide, Premium

75.0

25.0

1.0

If unsure of slide part number, refer to "generic" slide diagram below for generalguidance (images not to scale). Diagrams for verifying that tissue sections are placed in
the allowable area can also be found in the Visium CytAssist Quick Reference Cards Accessory Kit (Document CG000548). The diagrams demonstrate allowable areas thatare far enough away from frosted sections to not interfere with gasket closure during
the CytAssist assay. Frosted sections include the opaque area of the slide as well as any
etching on the slide.
While slides are specified as being 25 mm x 75 mm, manufacturing tolerances may leadto dimensions that are too small or large to be compatible with 10x Genomics products.
Tissue slide dimensions must be within 24.8 mm - 25.3 mm in width and 74.4 mm - 76.2
mm in length to fit the Visium CytAssist Tissue Slide Cassette.
Minimum slide dimensions: 24.8 x 74.4 mm

15 mm

15 mm

19 mm

FrostedArea

5

Generic

5

+

+

+

5

Epredia Shandon
ColorFrost Plus+VWR SuperFrost
Plus

5

5

15 mm

5

15 mm

15 mm

5

15 mm

5

15 mm

Tissueoutsidethis region
cannot beanalyzed

Frosted
Area

22 mm

15 mm

Frosted
Area

19 mm

Maximum slide dimensions: 25.3 x 76.2 mm

Sigma-Aldrich
Poly Prep

FisherbrandSuperFrost Plus

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5Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

1. Tissue Fixation & Embedding
Overview
This chapter provides guidance on fixing,
cryopreserving, and embedding of tissues. Freshly
obtained tissue samples are fixed in aparaformaldehyde (PFA) solution to preserve
tissues in their current state. Fixed tissues are thentransferred into a cryopreservant sucrose solution
to prevent the formation of ice crystals in thetissues. Once fixed and cryopreserved, tissue
samples are embedded in a freezing andembedding compound, Optimal Cutting
Temperature (OCT), to preserve the structure of thetissue and to provide structural support during
cryosectioning. Other methods of freezing andembedding for fixed frozen samples have not beenvalidated.

OCT embedding of the tissue offers the following
advantages:• Preserves the structure of the tissue and provides
structural support during cryosectioning.
• Maintains an optimal temperature duringsectioning, thus leading to smooth sections.

Tissue Fixation & Embedding
FreshTissueTissueFixation

4% PFA/PBS

Tissue Fixation
Tissue blocks are fixed in cold 4% PFA solution(pH 7.4) until the tissues sink to the bottom of thesolution, approximately 12-16 hours. Tissue block
size can impact fixation time. The fixed tissue
blocks are then cryopreserved upon placement in
30% sucrose solution until the tissues sink to thebottom of the solution, approximately 6-12 hours.
Ensuring that tissues are fully submerged in both
fixative and cryopreservant solutions is critical for
success.

Tissue Freezing & Embedding

30% sucrose/PBS

TissueFreezing &Embedding

OCT
must be frozen before
proceeding to next step

After fixation, and prior to cryosectioning, tissue
blocks are simultaneously frozen and embedded in
OCT utilizing an isopentane bath or powdered dry
ice.

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6Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

1.1 Specific Reagents and Consumables		

Paraformaldehyde should always be used with adequate ventilation in a fume hood.
Follow appropriate institutional regulations for handling and disposal of hazardous
waste.
Tissue Fixation
Item

Alternatives/Options

Paraformaldehyde

Paraformaldehyde
Paraformaldehyde, 16% w/v aq. solution, methanol
freePhosphate-Buffered Saline, 1X without calcium andmagnesium, pH 7.4

1X PBS
SucroseHCl
50-ml Centrifuge
Tubes
Petri Dishes
Filters

ForcepsSpatulaBeakerVendorPart
NumberMillipore Sigma 158127
Alfa Aesar

50-00-0Corning

21-040-CV

SucroseHydrochloric Acid Solution, 1 N
Or any equivalent HCl

Millipore Sigma S0389

50 mL centrifuge tubes

Millipore Sigma CLS430829

CytoOne Dishes

USA ScientificNalgene Rapid-Flow Sterile Disposable Filter Units
with PES Membranes

Thermo Fisher
Scientific
VWR

Syringe Filter, 0.22µm, sterile
Specimen Forceps, Straight, 203 mm (8”)

Honeywell

35328

CC76823359
565-0020
76479-024

VWR

82027-436

Specimen Forceps, Straight, 152 mm (6”)

VWR

82027-438

Round/Tapered Spatula, Stainless Steel

VWR
FisherScientific82027-490

Fisherbrand Polypropylene Disposable Beakers

FB0129111

Tissue Freezing & Embedding
Item

Alternatives/Options

Vendor

OCT

TissueTek O.C.T. Compound

VWR

Part
Number
25608-930

Molds

Disposable Based Molds (15 x 15 mm)
Dependent on the tissue size

VWR

60872-488

BeakerForceps
Cryovial
Additional Materials

Fisherbrand Polypropylene Disposable Beakers
Specimen Forceps, Straight, 203 mm (8”)
WHEATON 5 ml CryoELITE Tissue Vial

FisherScientific
VWR
Wheaton

FB0129111
82027-436
W985100

Dry ice--

pH Meter--Mortar and Pestle--

Rocker--10xgenomics.com

7Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

1.2 Tissue Fixation
		
		
		

Prepare solutions before harvesting of tissue. Large volumes of 4% PFA and 30% sucrose 		
solutions may be prepared and stored ahead of time. Store 4% PFA at 4°C and 			
30% sucrose at room temperature.
Items

Preparation & Handling

Prepare1X PBS

Pre-chill large volume of 1X PBS on wet ice or at 4°C prior touse.
Following preparation, fill a 50-ml centrifuge tube with 40 ml
cold 1X PBS. Prepare one centrifuge tube per tissue. Maintainon ice. Return 1X PBS to wet ice in between use.

4% PFA/PBS Solution

Weigh 4 g paraformaldehyde in chemical hood. Add 1X PBS
to volume of 99 ml. Slowly heat while stirring (DO NOT heat
>60°C). Cool to room temperature. Adjust to pH 7.4 with HCl.
Add 1X PBS to final volume of 100 ml. If prepared ahead of time,
store at 4°C.
Alternatively, use commercial 16% paraformaldehyde and dilute
to 4% concentration using 1X PBS.
Following preparation, fill a 50-ml centrifuge tube with 40 ml
4% PFA solution. Prepare one centrifuge tube per tissue.
Maintain on wet ice.

30% Sucrose/PBS Solution

Weigh 30 g sucrose. Add 1X PBS for final volume of 100 ml.
Vortex briefly. Filter using a sterile 0.22 µm filter. If preparedahead of time, store at room temperature.
Following preparation, fill a 50-ml centrifuge tube with 40 ml
30% sucrose solution. Prepare one centrifuge tube per tissue.
Maintain at room temperature.

a. Fill bottom of petri dish with cold 1X PBS. Transfer freshly obtained tissue to petri
dish using forceps or spatula.

			Work quickly between tissue harvest and fixation steps.
b. Rinse tissue using a pipette with cold 1X PBS to remove any residual blood.

Tissue Rinse

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8Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

c. Using either forceps or a spatula, transfer the tissue into a 50-ml centrifuge tube
containing freshly prepared, cold 4% PFA/PBS solution.
d. Gently agitate the centrifuge tube containing tissue on a rocker at the lowest setting
until tissue sinks to the bottom of the tube, approximately 12-16 hrs at 4°C.

			After 12-16 hrs, take tube off rocker and check that tissue has sunk to tube bottom. If not, 					check every 2-3 hrs for up to 24 hrs.
						It is essential that the tissue sinks to the bottom of the solution, indicating proper fixation. 		
Under-fixation can lead to issues with tissue integrity.
Tissue Fixation
Agitation on Rocker

Fully Sunk Tissuee. Using either forceps or a spatula, transfer the tissue into a 50-ml centrifuge tube
containing cold 1X PBS.
f.

Incubate for 1 min at room temperature.

g. Carefully discard cold 1X PBS, ensuring the tissue block stays at the tube bottom.
h. Repeat cold 1X PBS wash two more times.
			Discard 1X PBS

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9Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev Ai.

Using either forceps or a spatula, transfer the tissue into a 50-ml centrifuge tube
containing 30% sucrose/PBS solution and incubate until the tissue sinks to thebottom of the tube, approximately 6-12 h at 4°C.

			After 6 hrs, check that the tissue has sunk to tube bottom. If not, check every 2-3 hrs for up to 					12 hrs.
Tissue Cryopreservation
Transfer Tissuej.

Fully Sunk Tissue

Proceed immediately to the next step (Tissue Freezing & Embedding).

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10Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

1.3 Tissue Freezing & Embedding
Items
PreparePreparation & Handling

Fill two-thirds of a polypropylene beaker with isopentane
Isopentane bath (preferred method as (sufficient to fully submerge the cryomold) and place in dry
more rapidly freezes tissue)
ice (same level as isopentane) to allow sufficient contact.
Incubate for 15 min.
Powdered dry ice (alternative to
Use a mortar and pestle to prepare powdered dry ice.
isopentane bath)Pre-cooled OCT
Place OCT on wet ice for ≥30 min.
Pre-cooled forceps

Place forceps in dry ice for ≥30 min.

Pre-cooled cryovial or resealable bag
Confirm
Cryomold

Place cryovial or resealable bag on wet ice for ≥30 min.
The cryomold used for embedding should be of appropriate
size to fit the tissue sample.

Tissue Freezing & Embedding
Isopentane bath setup

a. Label an appropriately sized cryomold to mark the orientation of the tissue and placeat room temperature.

Label the cryomold before adding OCT and tissue. The OCT will quickly turn white once frozen,
making it hard to determine tissue orientation later.
b. Fill the cryomold with pre-cooled OCT without introducing bubbles.
c. Using pre-cooled forceps, place the tissue into the OCT, covering any exposed surfaces
with additional OCT. Confirm there are no bubbles, especially near the tissue.
d. Immediately place the cryomold containing tissue and OCT fully submerged in theisopentane bath or on powdered dry ice.
e. Wait until the OCT is completely frozen.
STOP

f.

Store the OCT embedded tissue block in a sealed container at –80°C for long-term
storage or immediately proceed to Cryosectioning & Section Placement.

A pre-cooled, WHEATON CryoELITE cryovial or a resealable bag should be used for storing the tissue
block. Remove the tissue block from the cryomold and if needed, trim it using a razor blade to fit into
the cryovial.
Failure to use a sealed container for storage may dehydrate and damage the tissue.

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11Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Tissue Freezing & Embedding
Isopentane bathTissue in OCT

Submerge into BathAfter Embedding

Powdered dry ice
Tissue in OCT

After Embedding

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12Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

2. Cryosectioning & Section Placement
2.0 Overview
This chapter provides guidance on cryosectioningof the OCT embedded tissue and placement of thetissue sections on blank slides.

Cryosectioning

Cryosectioning

OCT embedded tissue blocks are removed from the–80°C storage and cryosectioned in a cryostat togenerate sections for blank slides while keeping
samples in a cold environment.

RNA Quality Assessment
RNA quality of the tissue is assessed by calculating
DV200 of RNA extracted from OCT embedded fixedfrozen tissue blocks. Refer to RNA Quality
Assessment for details.

SectionPlacement

Section Placement
Tissue sections are placed within the allowablearea on compatible tissue slides. Placing only one
section per slide is recommended.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

2.1 Specific Reagents & Consumables
Cryosectioning
Item

Alternatives/Options

VendorPart
NumberOCT
50-ml Centrifuge
Tubes

TissueTek O.C.T. Compound

VWR

25608-930

50 mL centrifuge tubes

Millipore Sigma

CLS430829

Brushes

Magnetic Brush, big

VWR

334172

Brush, small beveled

VWR

14071425

Flat cryostat brush, 10 mm

Fisher Scientific14-071-00

Cryostat

CryoStar NX70 Cryostat
Vacutome, Low Profile Blade Carrier

Thermo Fisher
Scientific957020Specimen Chuck

Thermo Scientific CryoStar NX70 Specimen Chuck Fisher Scientific14-071-413

Slide Mailer

Simport Scientific LockMailer Tamper Evident
Slide Mailer(Alternatively, use a 50-ml centrifuge tube)

Fisher Scientific22-038-399

MX35 Ultra Microtome Blade
Low Profile

Fisher Scientific3051835

Glass Anti-Roll Plate

Fisher Scientific

A78930200

Epredia Shandon ColorFrost Plus Slides

Fisher Scientific6776214

Fisherbrand Superfrost Plus Microscope Slides

Fisher ScientificPoly-Prep Slides

Millipore Sigma

12-550-15
P0425

VWR Superfrost Plus Micro Slides

VWR

48311-703

Microtome Blade
Glass Anti-RollPlate, Optional
Blank Slides

Additional Materials
Forceps--

Dry ice--

Cryostat Chamber Specifications
This protocol describes the use of a Cryostar NX70 Cryostat with specific capabilities.
Alternatively, use a different cryostat with the following features.
Function

Notes

Main Cryochamber

Maintains stable temperatures from –10°C to –20°C

Cryostat Blade

Separate and adjustable temperature control
Maintains stable temperatures from –35°C to –5°C

Specimen Head

Separate and adjustable temperature control
Maintains stable temperatures from –50°C to +10°C
X-axis and Y-axis adjustment

Blade Holder Base

Adjustable cutting angle
Adjustable blade position
Section thickness 10-50 µm

Cryobar

Rapid cooling
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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

2.2 Cryosectioning
Items

Preparation & Handling

AdjustTurn cryostat on to pre-cool chamber. Recommended sectioning
Cryostat temperature temperature setting is –10°C to –20°C for cryostat blade and –10°C tosettings–20°C for the specimen head. Follow manufacturer's manual for detailed
operations.
Equilibrate
Blank slides to thecryostat chamber
temperature
OCT embedded
tissue block tocryostat chamber
temperature

Slides should be cooled down to cryostat temperature for ≥30 min. Warm slides
will lead to quick melting of the sections and degradation of RNA.
Freshly prepared or OCT embedded tissue block stored at –80°C must be
equilibrated to cryostat chamber temperature for at least 30 min before
sectioning. If the tissue block is too cold, it will lead to section cracking. If thetissue block is too warm, it will lead to section compression or crumpling.

OCT Embedded Tissue Block Mounting

a

b

c

d

Mount OCT Embedded Tissue Block on the Specimen Stage:a. Fill the specimen stage (chuck) with OCT.
b. Place the OCT embedded tissue block on the stage with the cutting surface facing
away from the stagec. Place the stage and the tissue block on the cryobar inside the cryostat chamber.
d. Allow the OCT and the tissue block to freeze and adhere to the specimen stage.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Exposing the Tissue:e. Once frozen, install the stage with the tissue block on to the specimen head of thecryostat and start sectioning to remove excess OCT.

Install Block onto Specimen Headf.

Sectioning conditions vary across different tissues and cryostats. Follow
manufacturer’s recommendation for cryosectioning.

g. Continue sectioning until the tissue is visible.
h. Acquiring sections from the tissue block for RNA quality assessment isrecommended. This should be done prior to placement of tissue onto blank slides.
Refer to step 2.4 RNA Quality Assessment for more information.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

2.3 Section Placement
Items

Preparation & Handling

Confirm
Section thickness setting
Anti-roll plate is in place, (Optional)

Recommended section thickness is 10 µm, but tissue section
thicknesses of 10−20 µm have been validated with the assay.
Anti-roll plate prevents rolling of tissue sections. Optimize
the position of anti-roll plate based on the tissue block
size. If possible, adjust the position of anti-roll plate before
reaching area of interest.

Specimen head temperature

Confirm the temperature of the specimen head that has
been optimized based upon tissue type. If the sections
appear cracked, the specimen head is too cold. If thesections appear crumpled, the specimen head is too warm.
Adjust temperature accordingly.

Slide electrostatic discharge

Validated, charged slides may improve tissue adhesion andare recommended for use in this Demonstrated Protocol.
If using a non-charged slide, prior to collecting the tissue
section, ensure that the slide has undergone electrostatic
discharge with a common metal within the cryochamber
to minimize the negatively-charged tissue section from
repelling. Allow slides to cool within the cryochamber for
5 minutes before mounting of the section.

metal surface

Slide storage

Pre-cool slide mailer or 50-ml centrifuge tube to cryostat
temperature for 10-15 minutes before use. If using centrifuge
tubes, store only one slide per tube.

Practice
Section placement on a
blank slide

Draw a representative allowable area on a 75 x 25 x 1 mmblank slide. Optimize section quality and practice section
placement within the allowable area before working withexperimental blocks. Refer to Determining Allowable Area.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Acquire Section and Place on Blank Slidea. Once desired tissue section is obtained, carefully
flatten it out by gently touching the surrounding
OCT with cryostat brushes.

Flatten the Section

b. Place the section within the allowable area on the
pre-equilibrated blank slide by gently touching thesection with the front of the slide.

DO NOT place sections on a room temperature slide.
Slide should be equilibrated to cryostat chamber.
c. Immediately place a finger on the backside of theslide for a few seconds to allow the section toadhere to the slide.

Ensure that the entire tissue section is fully adhered
to the slide and the slide is inside the cryostat chamber
throughout section placement.

Transfer the Section

DO NOT remove the slide from the cryostat chamber
at any point during sectioning and tissue placement.
d. Immediately place the slide with tissue section
on the cryobar to freeze the section. Continuetransferring sections on remaining slides.
e. Transfer the slides containing tissue sections to apre-cooled slide mailer. Alternatively, use a precooled 50-ml centrifuge tube.

Adhere the Section

If using 50-ml centrifuge tube, store slides individually.
If using an unsealed slide mailer, store in a secondary
sealed container, such as a resealable bag.
f.

STOP

Transfer slides in slide mailer or centrifuge tube
on dry ice.

g. Store slides in sealed slide mailer orcentrifuge tube at –80°C for up to 2 months
or immediately proceed to Visium CytAssist
Spatial Gene Expression for Fixed Frozen
Tissues – Rehydration, H&E Staining, Imaging& Decrosslinking Demonstrated Protocol(CG000662).

Immediately place the slide on thecryobar to allow section to freeze

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Leftover Tissue Block Storage:• Remove leftover tissue block attached to the specimen stage from the cryostat's
specimen head and place onto cryobar.
• Cover the exposed tissue with a thin layer of pre-cooled OCT and allow to freeze.

• The frozen tissue block can be stored attached to the specimen stage in a sealedcontainer at –80°C. To separate the frozen tissue block from the stage, lift the tissue
block and the stage from the cryobar and lightly warm the stage with hands or an
aluminum block at room temperature.
DO NOT let the block and tissue fully melt, as this will severely damage the tissue.
Separation of the tissue block from the specimen stage is optional.
• Immediately place the tissue block in dry ice. Ensure that the melted areas haverefrozen.
• Store in a sealed container at –80°C for long-term storage.

Slide Shipping:• Place slides in a slide mailer and keep cold. If multiple slides are being shipped,
ensure that there is sufficient space in between the slides to avoid contact. If placing aslide in the last slot, ensure slide does not face outward.

• Place the mailer in a tightly sealed secondary container to limit exposure and keep
cold.
• Samples can be shipped overnight in dry ice, provided there is enough dry ice toaccount for transit and delivery times.
• Refer to the local institution or delivery service for detailed instructions on shipping
samples in dry ice.
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19Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

2.4 RNA Quality Assessment
This section provides guidance on assessing the quality of OCT embedded fixed frozen tissue blocks by
calculating its DV200 score. This protocol was optimized using samples with DV200 scores ≥50%.
For RNA Quality Assessment
Item
RNA Isolation
RNase Decontamination
Solution
Nuclease-free Water

Alternatives/Options
CELLDATA RNAstorm FFPE RNA Extraction Kit

Vendor
Biotium

RNaseZap RNase Decontamination Solution

Thermo Fisher Scientific AM9780

Nuclease-free Water (not DEPC-Treated)
Ethyl Alcohol, 200 Proof
Ethanol
Ethanol absolute ≥99.5%, TechniSolv, pure (Europe Only)
1.5-ml Centrifuge Tubes
DNA LoBind Tubes, 1.5 ml
0.2 ml PCR 8-Tube Strips
PCR Tubes 0.2 ml 8-tube strips
TempAssure PCR 8-tube strip
Choose either Eppendorf, USA
MicroAmp 8-TubeStrip, 0.2 ml
Scientific, or Thermo Fisher
MicroAmp 8-cap Strip, clear
Scientific PCR 8-tube strips
Nanodrop/Qubit Fluorometer Nanodrop 2000c Spectrophotometers
Or any equivalent Nanodrop
Alternative to Qubit Fluorometer
Qubit RNA BR Assay Kit

Bioanalyzer/TapeStation
Choose Bioanalyzer or
TapeStation based on
availability & preference.

Part Number
CD501

Thermo Fisher Scientific
Millipore Sigma
VWR
Eppendorf
Eppendorf
USA Scientific
Thermo Fisher Scientific
Thermo Fisher ScientificAM9937
E7023
83813.360DP
022431021
951010022
1402-4700
N8020580
N8010535

Thermo Fisher Scientific ND-2000C
Thermo Fisher Scientific Q10210

Qubit Assay Tubes

Thermo Fisher Scientific Q32856

Qubit 4 Fluorometer
2100 Bioanalyzer Laptop Bundle
4200 TapeStation
RNA ScreenTape
RNA ScreenTape Ladder
RNA ScreenTape Sample Buffer

Thermo Fisher Scientific
Agilent
Agilent
Agilent
Agilent
Agilent

Q33238G2953CA
G2991AA
5067-5576
5067-5577
5067-5578

Additional Materials
Razor blades
Forceps
Dry ice
Wet ice--

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

RNA Quality Assessment
Pre-cool microcentrifuge tube, cooling block, and forceps in cryostat chamber or at–20°C to prevent premature melting of tissue sections.
Collect tissue sections

Analyze RNA quality

RNA isolation

DV200 ≥50%

Proceed to Visium
CytAssist Spatial
Protocolsa. Cryosection 20–30 mg of tissue sections from the OCT embedded tissue block(~4 sections at 25 µm thickness). If tissues contain extensive connective or adipose
tissue, cryosection up to 50 mg of tissue.
b. If OCT is excessive (�1 mm surrounding the tissue), remove excess OCT with a razor
blade or with cooled forceps.
c. Using the cooled forceps, transfer sections to a pre-cooled microcentrifuge tube.
d. Place the pre-cooled microcentrifuge tube containing sections on dry ice. Store at–80°C or proceed to RNA extraction.

			DO NOT allow samples to melt, as this will lead to degradation of RNA and a poor DV200 score.
e. Proceed with RNA isolation starting at step A5 using the Biotium CELLDATA
RNAstorm FFPE RNA Extraction Kit. After RNA isolation, place samples on wet ice.
			Fixed frozen tissue is not paraffin embedded and therefore should not undergo the 						

deparaffinization steps as described in steps A1-A4.
f.

Store purified RNA at –80°C for long-term storage or immediately proceed toDV200 calculation using either BioAnalyzer, TapeStation, or ScreenTape reagents.
Follow manufacturer's instructions (Agilent) for DV200 calculation.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

DV200 Performance and Recommendations
DV200 is a broad measurement of RNA quality and is influenced by factors including:• Tissue block age, type, and composition• Region selected for RNA extraction• Presence of diseased or necrotic regions• Depth of section• Fixation method• Miscellaneous upstream tissue handling and processing
A DV200 score of ≥50% is recommended for the Visium CytAssist Spatial Gene
Expression for Fixed Frozen workflow.

Example DV200 Traces
Bioanalyzer traces of RNA extracted from different tissue types for which DV200 was
calculated are shown below.

Bioanalyzer Traces for Mouse Brain and Mouse Kidney Samples
5,000
4,000

500
1,000
2,000
4,000
6,000

[nt]

500
1,000
2,000
4,000
6,000

0

200

3,000
2,000
1,000
25

[Normalized FU]

A. DV200 = 56%

[nt]

5,000
4,000

0

200

3,000
2,000
1,000
25

[Normalized FU]

B. DV200 = 82%

Samples displayed are from Mouse Brain (DV200=56%) (A) and Mouse Kidney (DV200=82)% (B). The Biotium
CELLDATA RNAstorm FFPE RNA Extraction Kit was used for RNA isolation, while the TapeStation RNA ScreenTape Kit
was used for DV200 calculation.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Troubleshooting
Incorrect Placement of Tissue Sections

Folded tissue
sectionOverlapping
sections

Cracking of Tissue Sections
Causes• A tissue block that is too cold can crack while sectioning.
• Sectioning too quickly can lead to cracking.
Troubleshooting• Reduce sectioning speed and/or increase temperature to
reduce cracking.
Impact of Cryostat Specimen Head Temperatures on Tissue Tearing
-10°C
-14°C

-20°C

-30°C

Normal Section
The temperatures shown are representative. Ideal speciman head temperature can vary between tissue types and blocks.

Under-Fixation of Tissue Sections
Ensure tissue block has dropped to tube bottom as recommended. Characteristics of an under-fixed tissue may include:• Pale or translucent appearance.
• Lack of structure and gelatinous to touch.
• Shrinkage of tissue.

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Demonstrated Protocol | Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue Preparation Guide CG000663 | Rev A

Document Revision Summary
Document Number

CG000663

Title

Visium CytAssist Spatial Gene Expression for Fixed Frozen - Tissue
Preparation Guide

Revision

Rev A

Revision Date

March 2023

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