CG000636 | Rev A

DEMONSTRATED PROTOCOL

Visium CytAssist Spatial Gene Expression for
Fresh Frozen – Tissue Preparation Guide
Introduction
The Visium CytAssist Spatial Gene Expression Solution analyzes mRNA from tissue sections on blankslides. Proper tissue handling and preparation techniques preserve the morphological quality of the tissue
sections and the integrity of mRNA transcripts. This is critical for downstream library preparation andgeneration 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 blank slides before and after cryosectioning.
• Freezing and embedding tissue samples prior to cryosectioning.
• Cryosectioning of tissue samples and placement of sections on blank slides.
• Assessment of RNA quality.
• Tested blank slides compatible with the Visium CytAssist Spatial assay.

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 specialized tissues,
such as tissue with high fat content.

12Introduction
Tips & Best Practices

45

Determining Allowable Area
Visium CytAssist Tested Slides

The slides prepared using the Tissue Preparation
Guide can be used with:• Visium CytAssist Spatial Gene Expression for
Fresh Frozen – Methanol Fixation, H&E Staining,
Imaging & Destaining Demonstrated Protocol(CG000614)• Visium CytAssist Spatial Gene Expression User
Guide (CG000495) Rev C or later

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1. Tissue Freezing & Embedding

78
9

1.1 Reagents & Consumables
1.2 Tissue Freezing
1.3 Frozen Tissue Embedding10

2. Cryosectioning & Section Placement

11
12
14
16
16
17

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

20

Troubleshooting

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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 reagentsand consumables.

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

• Cryosectioning temperatures impact tissue section
integrity. A temperature setting of –20°C for blade
and –10°C for the specimen head is recommended.

• Prior to section placement, draw an outline of
the allowable area on the back of the blank slide
to ensure downstream compatibility with the
Visium CytAssist Tissue Slide Cassette and Visium CytAssist instrument (Refer to Determining
Allowable Area). Drawing should be removed
after tissue placement.

• The temperature settings depend upon the local
conditions, tissue types, and the cryostat used.
Settings should be optimized based on the quality of resulting tissue sections.

• If working with multiple sections on multiple tissue slides, ensure that sections are placed in thesame location on the tissue slides for improved
imaging efficiency.

• During prolonged sectioning periods, allow thecryostat temperature to equilibrate by briefly
closing the chamber.

• If placing multiple sections on a blank slide:

Cryosectioning Temperature

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

• Ensure that tissue sections and associated
OCT do not overlap with other tissue sections
and associated OCT.
• Ensure sections previously placed on the slide
are not thawed while placing other sections.
• Ensure all sections are placed during thesame sectioning event.
• Only one section from each tissue slide
should be used for each Visium CytAssist
Spatial Gene Expression Slide Capture Area.

Section Thickness• Recommended section thickness for most tissue
types is 10 µm, but tissues from 10−20 µm arecompatible with the assay. Tissues with higher fat
content (e.g., breast tissue) may require sections
closer to 20 µm.
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Practice Section Placement• Practice correct section placement using non-experimental blocks.
• Practicing section placement also allows fordetermining an ideal tissue thickness for the tissue 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.

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.

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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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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 general guidance (images not to scale). Diagrams for verifying that tissue sections are placed in theallowable area can also be found in the Visium CytAssist Quick Reference Cards - Accessory Kit (Document CG000548). The diagrams demonstrate allowable areas that are 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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1. Tissue Freezing & Embedding
Overview
This chapter provides guidance on tissue freezing
and embedding. Freshly obtained tissue samples
must be snap frozen to prevent RNA degradation
and avoid ice crystal formation, which can lead tomorphological damage to the tissue. Once frozen,
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 have not been validated.

Tissue Freezing
A bath of isopentane and liquid nitrogen is used tofreeze freshly obtained tissue. Tissue should not be
placed directly in liquid nitrogen as thetemperature difference may cause boiling on thesurface of the tissues, leading to air pockets anduneven freezing. This may crack andmorphologically damage the tissue.

Frozen Tissue Embedding
After freezing and prior to cryosectioning, tissue
samples are embedded in OCT on dry ice or in
isopentane. Unlike paraffin or resin-based
embedding techniques, OCT does not interact withproteins and other molecules that impact
antigenicity.

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 during sectioning, thus leading to smooth sections.
• Compatible with multiple staining procedures
due to its water solubility.

Freezing and Embedding
FreshTissueTissueFreezing

Isopentane
LiquidNitrogen

Frozen Tissue
Embedding

OCT
must be frozen before
proceeding to next step

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1.1 Specific Reagents and Consumables
Tissue Freezing
Item

Alternatives/Options

VendorPart Number

Isopentane

Isopentane (2-Methylbutane)

Millipore Sigma270342ForcepsSpecimen Forceps, Straight, 203 mm (8”)

VWR

82027-436

Specimen Forceps, Straight, 152 mm (6”)

VWR

82027-438

SpatulaRound/Tapered Spatula, Stainless Steel

VWR

82027-490

Beaker

Stainless Steel Beaker (250 ml)

VWR

89075-592

Cryovial

WHEATON 5 ml CryoELITE Tissue Vial

WheatonW985100

MorterLiquid Nitrogen Cooled Mini Morter, 1.5 mL Capacity
Remove 1.5 mL tube holder prior to use.
Choose appropriate dewar size based on the size ofthe steel beaker used.

Grainger

21TT88Frozen Tissue Embedding
Item

Alternatives/Options

VendorPart Number

OCT

TissueTek O.C.T. Compound

VWR

25608-930

Molds

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

VWR

60872-488

Razor blades--

Liquid Nitrogen--

Dry ice--

Additional Materials

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1.2 Tissue Freezing
Items

Preparation & Handling

PrepareIsopentane and liquid nitrogen bathOR dry ice

Fill two-thirds of a metal beaker with isopentane (sufficient
to fully submerge the tissue) and place in a liquid nitrogen
dewar (same level as isopentane) to allow sufficient contact.
Incubate 5-10 min.
Isopentane will slowly freeze if left too long in the liquid
nitrogen bath. When this occurs, briefly remove the metal
beaker containing isopentane from the liquid nitrogen bath.
Once thawed, place the beaker back in the liquid nitrogen bath.
DO NOT use the same isopentane bath for different
tissue types.

TissueUsing a rolled up laboratory wipe, absorb excess blood orsolution from the surface of the tissue to limit ice crystal
formation. Tissue cracking may occur if tissue has been left
in buffer for extended periods.

Pre-cooled Cryovial

Pre-cool a WHEATON CryoELITE cryovial on dry ice.

a. Using either forceps or a spatula, lower the tissue into the isopentane until fully
submerged. Keep tissue submerged for ~1 min or until frozen. The freezing time
may vary based upon the tissue type and size.
b. Once frozen, transfer the tissue to the pre-cooled WHEATON CryoELITE
cryovial on dry ice.
STOP

c. Store frozen tissue at –80°C for long-term storage or immediately proceed to
the next step (Frozen Tissue Embedding).

To prevent evaporation and dehydration, the snap-frozen tissue block must be stored in a sealedcontainer.

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1.3 Frozen Tissue Embedding
Items
PreparePowdered dry ice

Preparation & Handling
Use a mortar and pestle to prepare powdered dry ice.
Fill two-thirds of a polypropylene beaker with isopentane(sufficient to fully submerge the cryomold) and place in dry
ice (same level as isopentane) to allow sufficient contact.
Incubate for 15 min.
Place OCT on wet ice for ≥30 min.

Isopentane bath (alternative topowdered dry ice)
Pre-cooled OCT
Pre-cooled forceps
Confirm

Place forceps in dry ice for ≥30 min.
The cryomold used for embedding should be of appropriate
size to fit the tissue sample.

Cryomolda. 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. Remove cryovial containing frozen tissue from –80°C (if tissue was stored after step1.2) and transfer in dry ice.
c. Fill the cryomold with pre-cooled OCT without introducing bubbles.
d. Using pre-cooled forceps, place the frozen tissue into the OCT, covering any exposed
surfaces with additional OCT. Confirm there are no bubbles, especially near the tissue.
e. Immediately place the cryomold containing tissue and OCT on powdered dry ice orisopentane bath. If using isopentane, lower the cryomold containing tissue into theisopentane without fully submerging.
f.
STOP

Wait until the OCT is completely frozen.

g. 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 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.
Frozen Tissue Embedding
Tissue in OCT

After Embedding

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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
It is recommended to assess RNA quality of thetissue block at this stage by calculating RNA
Integrity Number (RIN) of freshly collected tissue
sections. RNA quality assessment should be done
before placing the tissue sections on blank slides.
Use sections from the tissue block.

SectionPlacement

Refer to RNA Quality Assessment for details. RIN
should be ≥ 4. Low RIN scores do not necessarily
result in poor data, but high scores are more likely
to correlate with good data. Various factors could
lead to low RIN scores, such as specific tissue types,
diseased or necrotic tissues, ischemic tissue, andsuboptimal sample preparation and handling.

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

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2.1 Specific Reagents & Consumables
Cryosectioning
Item

Alternatives/Options

VendorPart
Number

OCT

TissueTek O.C.T. Compound

VWR

25608-930

Centrifuge Tubes Sterile Centrifuge Tubes with Flat Caps, 50 ml

VWR

82018-050

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
Scientific957020Speciman 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-RollPlate, Optional

Glass Anti-Roll Plate

Fisher Scientific

A78930200

Blank slides

Epredia Shandon ColorFrost Plus Slides

Fisher Scientific6776214

Fisherbrand Superfrost Plus Microscope Slides

Fisher Scientific12-550-15

Poly-Prep Slides

Millipore Sigma

P0425

VWR Superfrost Plus Micro Slides

VWR

48311-703

Microtome Blade

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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2.2 Cryosectioning
Items

Preparation & Handling

AdjustTurn cryostat on to pre-cool chamber. Recommended sectioning
Cryostat temperaturetemperature is –20°C for cryostat blade and –10°C for the specimen head.
settings
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.

Mount OCT Embedded Tissue Block on the Specimen Stage:
OCT Embedded Tissue Block Mounting

a

b

c

d

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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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 can be done prior to or after placement of tissue onto blankslides. Refer to step 2.4 RNA Quality Assessment for more information.

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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.
If the sections appear cracked, the specimen head is toocold. If the sections appear crumpled, the specimen head is
too warm. Adjust temperature accordingly.

Slide electrostatic discharge

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
min before mounting of the section.

metal surface

Slide storage

Pre-cool slide mailer or 50-ml centrifuge tube to cryostat
temperature for 10-15 min.

Practice
Section placement on a
blank slide

Create 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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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.
Immediately place the slide with tissue section
on the cryobar to freeze the section. Continuetransferring sections on remaining slides.
d. Transfer the slides containing tissue sections to apre-cooled slide mailer. Alternatively, use a precooled 50-ml centrifuge tube.

Adhere the Section

Store slides individually (one slide per 50-ml centrifuge
tube, if using) in a sealed container. If using an unsealed
slide mailer, store in a secondary sealed container, suchas a resealable bag.
e. Transfer slides within slide mailer or centrifuge
tube to dry ice.
STOP

f.

Store slides at –80°C for up to 2 months or
immediately proceed to Visium CytAssist
Spatial Gene Expression for Fresh Frozen –Fixation, H&E Staining, Imaging & Destaining
Demonstrated Protocol (CG000614).

Immediately place the slide on thecryobar to allow section to freeze

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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 sealed container at –80°C. To separate the frozen tissue block from the stage, lift the tissue blockand 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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2.4 RNA Quality Assessment
This section provides guidance on assessing the quality of the OCT embedded tissue
blocks by calculating its RNA Integrity Number (RIN).
RNA Extraction
Item

Alternatives/Options

VendorPart Number

RNA Isolation Kit

RNeasy Mini Kit (50)Refer to Purification of Total RNA from Animal
Tissues in RNeasy Mini handbook
QIAshredder (50), Optional

Qiagen74104

Qiagen79654

CELLDATA RNAstorm Fresh Cell and Tissue
RNA Isolation Kit

Biotium

CD504

Centrifuge Tubes

DNA LoBind Tubes, 1.5 ml

Eppendorf

022431021

2-Mercaptoethanol
Ethanol(if using CELLDATA
protocol)2-Mercaptoethanol

Millipore Sigma M6250-100ML

Ethyl Alcohol

Millipore Sigma M7023-500ML

Bioanalyzer/
Tapestation

RNA 6000 Pico Kit

Agilent

5067-1513

2100 Bioanalyzer Laptop Bundle

Agilent

G2953CA

Choose Bioanalyzer
or TapeStation
based on availability& preference.

4200 TapeStation

Agilent

G2991AAHigh Sensitivity RNA ScreenTape

Agilent

5067-5579

High Sensitivity RNA ScreenTape Ladder

Agilent

5067-5581

High Sensitivity RNA ScreenTape Sample Buffer Agilent

5067-5580

Additional Materials
Nuclease-free Water--

Razor blades--

Forceps--

Dry ice
Wet ice--

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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

RIN ≥ 4

Proceed to Visium
CytAssist Spatial
Protocolsa. Cryosection 20–30 mg of tissue sections from the OCT embedded tissue block (~4sections 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 todegradation of RNA and a poor RIN score.
e. Isolate RNA according to manufacturer's instructions. After RNA isolation, placesample on wet ice.
f.

Store purified RNA at –80°C for long-term storage or immediately proceed to RIN
calculation using either BioAnalyzer or TapeStation reagents. Follow manufacturer's
instructions for RIN calculation.

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RIN Trace Examples
This section contains example traces for RNA Integrity Number (RIN) assessment.

This protocol was optimized using samples with RIN ≥4.
TapeStation Traces for Mouse Brain and Human Breast Cancer Samples
Marker

18S Fragment

28S Fragment

Samples displayed above are from intact (mouse brain, yellow, RIN = 7; ) and degraded(human breast cancer, blue, RIN = 4.4) RNA. The CELLDATA RNAstorm Fresh Cell andTissue RNA Isolation KIt was used for RNA isolation, while the TapeStation HighSensitivity RNA Screentape kits was used for RIN calculation.

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Troubleshooting
Impact of Cryostat Specimen Head Temperatures on Tissue Tearing
-10°C
-14°C

-20°C

-30°C

Normal Section
Impact of Condensation on Tissue Sections

No Condensation. Intact tissue section.

Tissue degraded due to condensation. Always transportslides with tissue sections on dry ice to avoid condensation.
DO NOT leave slides at room temperature, especially withfresh sections as the resulting condensation will cause tissue
disintegration.

Incorrect Placement of Tissue Sections

Folded tissue
sectionOverlapping
sections

Practice correct section placement on blank slides before proceedingwith slides that will be used for the Visium CytAssist Spatial Assay.
See Visium CytAssist Tested Slides for templates.

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20

Demonstrated Protocol | Visium CytAssist for Fresh Frozen Tissue Preparation Guide

CG000636 | Rev A

Document Revision Summary
Document Number

CG000636

Title

Visium CytAssist Fresh Frozen Tissue Preparation Guide

Revision

Rev A

Revision Date

January 2023

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