Stereo-seq Sample Preparation,
Sectioning and Mounting Guide
for Formalin-fixed and Paraffinembedded (FFPE) Samples on
Stereo-seq Chip Slides
USER MANUAL

STUM_SP003

TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION
1.1. Introduction							1CHAPTER 2: SAMPLE PREPARATION
2.1. Reagent Preparation						32.2. Sample Requirements for Formalin-fixed and Paraffinembedded (FFPE) Tissue 					62.3.

Tissue Sectioning and Mounting				

9

APPENDIX
Appendix I: Shipping Preservation and Transportation

15

NOTE: Additional operation tips and guidance.
CRITICAL STEPS: Pay extra attention for these steps to avoid experimental setbacks or
problematic results.
QUALITY CHECK POINT
CAUTION: Proceed with extra care; improper handling or carelessness may cause
experimental failure or accidents.
STOP POINT: Here you may pause your experiment and store your sample.

CHAPTER 1
INTRODUCTION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

1.1. Introduction
The STOmics Stereo-seq Transcriptomics Set for FFPE is intended for generating aspatially-resolved total RNA library from formalin-fixed and paraffin-embedded (FFPE)
biological tissue sections that requires a Stereo-seq Chip Slide with intact tissue
sections as input. This guide provides general guidelines for properly performing tissue
sectioning and mounting to better preserve the morphological quality of the FFPE tissue
sections.
The Stereo-seq Chip Slides prepared in this guide are part of these products:
Stereo-seq Transcriptomics Set for FFPE (211SN114)

Stereo-seq Chip N Slide•

Includes 4 Stereo-seq Chip N Slides containing one Chip N (1cm*1cm) on each slide.

•

Stereo-seq Chip P Slides, Stereo-seq Chip T Slides, and Stereo-seq Chip N Slides aredifferentiated by a laser-engraved label at the end of the slide.

1

1. INTRODUCTION

CHAPTER 2
SAMPLE
PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

2.1. Reagent Preparation
Unless otherwise specified, nuclease-free water is used for all reagents being
prepared prior to this experiment.
Reagent Preparation Master Planning Table
Only the row highlighted in purple will be used in this guide. Please use thefollowing master planning table as your reference when planning for subsequent
experiments.

Prep
Day

Reagent

Workflow
Section

Day 1

400 mL 30%
ethanolTissueDilute anhydrous ethanol tosectioning and
30% using ddH2O.
mounting

Room
Temperature up
to 1 day

100 mL 96%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
96% using ddH2O.

Room
Temperature up
to 1 day

50 mL 90%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
90% using ddH2O.

Room
Temperature up
to 1 day

50 mL 80%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
80% using ddH2O.

Room
Temperature up
to 1 day

50 mL 70%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
70% using ddH2O.

Room
Temperature up
to 1 day

50 mL 50%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
50% using ddH2O.

50 mL 30%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
30% using ddH2O.

Day 2

Preparation Steps

Maintenance

Room
Temperature up
to 1 day
Room
Temperature up
to 1 dayHematoxylin(Solarbio,
H&E staining
G4470)Take it out of 4°C in advanceRoomand equilibrate to room
Temperature up
temperature at least 5 min
to 2 hrprior to use.

Eosin
Solution

Eosin was diluted with 70% Roomethanol at a 2:1 volume ratio. Temperature

H&E staining

50 mL 5X SSC H&E staining
200 mL 0.1X
SSC

H&E staining3

Room
Add 12.5 mL 20X SSC into 37.5
Temperature up
mL ddH2O and mix well.
to 1 week
Room
Add 1 mL 20X SSC into 199 mL
Temperature up
ddH2O and mix well.
to 1 week
2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User ManualRoom
Add 50 μL 20X SSC into 150 μL
Temperature up
200 μL 5X SSC ssDNA staining
ddH2O and mix well.
to 1 week

Day 2

50 mL 0.1X
SSC

ssDNA stainingRoom
Add 250 μL 20X SSC into 49.75
Temperature up
mL ddH2O and mix well.
to 1 week

FFPE
Mounting
Medium

H&e staining
and ssDNA
staining

Take it out of -20°C in advanceRoomand equilibrate to room
Temperaturetemperature until it thaws.

Fluorescent
Staining
SolutionRoom
Add 1 μL Qubit ssDNA
Temperature in
ssDNA staining Reagent and 10 μL RI into 189
the dark up to 1
μL 5X SSC and mix well.
hr

TE buffer(pH=9.0)

Take it out of 4°C in advanceRoomand equilibrate to room
H&E
Temperature up
decolorization temperature at least 5 min
to 2 hrprior to use.

FFPE
Decrosslinking
Reagent

Take it out of -20°C in
advance, equilibrate to room
Room
temperature until it thaws,
Decrosslinking
then mix well to ensure that Temperature
no white precipitates arevisible.

If white precipitates are visible in the reagent, dissolve them by heating
the buffer at > 50 °C and equilibrate to room temperature before mixing.

0.01N HCl

Permeabilization

Room
temperature for
Prepare at least 1 mL of 0.01N 48 hr (Storing
HCl per sample. Configure
longer than 48
HCl to 0.01N. Measure andhr will affectmake sure the pH = 2.
the desired
pH. Please use
WITHIN 48 hr ofpreparation)

ALWAYS use freshly prepared 0.01N HCl (pH = 2.0 ± 0.1). For pre-made 0.1N
HCl and newly purchased HCl, check the pH prior to the experiments.
10X
Permeabilization
Reagent
Stock
Solution

Permeabilization

Add 1 mL of freshly prepared
0.01N HCl to dissolve PR-20°C up to 1
Enzyme (red cap, in powder
monthform), and thoroughly mix thereagent through pipetting.

DO NOT vortex the permeabilization enzyme. Mix by pipette before using.
Aliquot this 10X stock solution to avoid freeze-thaw cycles.

4

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

1X
Permeabilization
Reagent
Solution

Permeabilization

Make 1X PR solution (200 μL /On ice until use,chip) by diluting 10X PR stock
up to 6 hrsolution with 0.01N HCl.

0.1X SSC

Permeabilization

Add 10 μL 20X SSC into 1990
μL nuclease-free water andmix well.

0.1X SSC(with 5% RI)

Permeabilization

Add 10 μL RI into 190 μL 0.1X
On ice until use
SSC and mix well

FFPE RT
Buffer Mix

Take it out of -20°C in
advance, equilibrate to room
temperature until it thaws,
RT and ligation
On ice until use
then mix well to ensure thatno white precipitates arevisible.

FFPE Dimer

RT and ligation

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

FFPE RT Oligo RT and ligation

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

cDNA ReleasecDNA Release
BufferTake it out in advance andheat the buffer for 5 min
at 55°C to dissolve theprecipitate. Equilibrate it toroom temperature prior to
use.

Room
temperature

Day 2

Room
Temperature up
to 1 week

If white precipitates are visible in the buffer, dissolve them by heating thebuffer at 55 °C again and equilibrate to room temperature before mixing.

Day 3

Magnetic
beads

cDNA
Purification

Take it out in advanceand equilibrate to room
temperature at least 30 min
prior to use.

10 mL 80%
Ethanol

cDNA
Purification

Room
Dilute 100% ethanol to 80%. temperature up
to 1 day

cDNA
cDNA
Amplification
Amplification
Mix
FFPE cDNA
Primer Mix

cDNA
Amplification

TE buffer, pH cDNA
8.0
Purification

54°C

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

Set it aside at room
temperature until use

Roomtemperature

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

2.2. Sample Requirements for Formalin-fixed and Paraffin-embedded (FFPE) Tissue
Sample Types
This set of kits can be used for samples from all common animals, including but notlimited to human, monkey, and mouse.

Sample Preparation•

Ensure that tissue samples are promptly fixed with 10% formalin or 4%paraformaldehyde (PFA) for 12 to 48 hr after sampling. It is preferable to use 10%
formalin for fixation.

•

To prevent drying, corrosion, and mildew, seal wax blocks properly.

•

When processing samples, it is important to maintain the original tissue structure.

•

Refer to your institution's pathology/histology labs for guidance on making paraffinblocks. This process requires adequate dehydration and wax immersion to produce
continuous slices without cracks or gaps (apart from the tissues themselves), ensuringthat the specimen remains embedded in the wax during sectioning.

•

Paraffin block samples can be sent directly. For additional details, please refer toAppendix I: Shipping Preservation and Transportation.

FFPE Sample RNA Quality Control (QC) by DV200 Value
Prepare the following:

Reagent

Preparation Steps

PurposeRPE buffer

Strictly follow the instructions from
Qiagen, and add ethanol in advanceRNA extraction

DNAse I

Strictly follow the instructions from
Qiagen. Dissolve the powder, aliquot the
RNA extraction
solution, then store it at -20°C . Take it out
in advance and keep it on ice until use.

It is recommended that you check the RNA quality (DV200 value) of a tissue sample
before proceeding to the Stereo-seq experiment. Total RNA can be extracted from 3-5
slices of 5 μm-thick FFPE sections (Deparaffinization Solution, Qiagen 19093 and RNeasy
FFPE Kit, Qiagen 73504) and the RNA can be stored at -80°C in a 1.5mL EP tube for up to 1
month . Refer to Figure 1 for examples of DV200 distribution ranges after quality control
of different RNA samples, using Agilent High-Sensitivity RNA Analysis Kit (Agilent 50671513).
It is strongly recommended that you proceed only with tissue samples with a
DV200 ≥ 30%.

6

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

DV200: 30%~50%

DV200: 50%~70%

7

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

DV200: >70%

Figure 1. Examples of DV200 distribution intervals after different RNA quality control

8

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

2.3. Tissue Sectioning and Mounting

For a demonstration video of the paraffin section mounting on the Stereo-seq Chip
Slide, refer to the link below or scan the QR code:
https://en.stomics.tech/resources/videos/list.html

30% Ethanol solution(300~400 mL)

Microscopic glass slides

Cardstock (cover stock) paper

Ice box and FFPE block

Histology brushes

Forceps

Microtome blades

Prepare the following:

Reagent

Preparation Steps

Purpose

400 mL
30% ethanol

Dilute 120 mL of anhydrous ethanol to 30% Paraffin blockusing 280mL ddH2O. Always use freshly
sectioning andprepared 30% ethanol.
mounting

This procedure should be carried out by technicians who are experienced inperforming paraffin sectioning.
a.

Make sure your water bath and baking machine have been turned on and set to 40°C
~ 48°C and 42°C, respectively.
If a different water bath temperature has been employed at your institution's
pathology/histology laboratories, always adhere to the established protocols.
If a PCR thermal cycler is used in place of a baking machine, place the PCR Adaptor
on the baking machine in advance, and set the PCR thermal cycler according to thefollowing incubation protocol.
9

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

Program selection: Incubate

Temperature

Time(Heated lid) 45°C

on

42°C

∞

42°C

3 hr

37°C

∞

b.

Prepare the microtome, histology brushes, forceps, new microtome blades, and acontainer filled with 300 ~ 400 mL of 30% ethanol.

c.

Place the paraffin block face down in an ice water mixture for 10 ~ 30 min, or cool
the tissue surface on a cooling platform for 5 ~ 10 min.

For tissues with high-fat content, such as breast tissue samples, freeze the FFPE
block at -20°C for 10 min before sectioning.
d. Take the Chip Slide out of the vacuum-sealed aluminum bag and record the Chip ID(SN) number located on the back side of the slide. Do not touch the front side of thechip.
After opening the bag, check all Stereo-seq Chip Slides in the slide container
and make sure they are oriented with the front sides facing up. The front sideof a chip has a shiny surface that contains DNB-probes for RNA capture. DO NOT
scratch the surface.

e.

Make sure the PCR thermal cycler has been turned on and set to 42°C.

f.

Equilibrate the Stereo-seq Chip Slide to room temperature for 1 min on thebenchtop, then rinse with 100 μL nuclease-free water twice with a pipette or rinse
the slide up and down twice in a 50 mL centrifuge tube with sufficient nuclease-freewater.

10

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

Store unused slides in the original packaging (first in the slide container andthen in the sealable aluminum bag) and keep sealed at -25°C ~ 8°C. KEEP thedesiccant in the aluminum bag.
g. Remove excess water from the chip by blowing gently with a power dust remover(MATIN, M-6318). Wipe off excess water from around the chip and on the slide with
dust-free paper.

h.

Only when the chip is completely dry and without wavy white stains is it ready fortissue mounting.

i.

Insert the paraffin block on the microtome and orient it such that the blade will cut
straight across the block. If the tissue is deeply embedded, approach the block withan old trimming blade and cut a few thin sections to ensure that the positioning iscorrect. Adjust if necessary.

j.

Trim the block to expose enough tissue surface from which a representative section
can be cut. Trimming is normally done at a thickness of 10~30 µm.

k.

Adjust the section thickness to 5 μm for regular tissue and 4 μm for high-fat-content
tissue to reduce the possibility of section detachment in subsequent operations.

l.

Discard the first section if the thickness does not meet the requirements. Select thedesired section during subsequent sectioning and use it for subsequent trimmingand mounting on the Stereo-seq Chip Slide. Using clean forceps, carefully transfer
the selected section onto cardstock paper for trimming.

11

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

To preserve adjacent sections, make a ribbon of three continuous sections. Use thefirst and third sections for subsequent H&E staining on microscopic glass slides.
Carefully separate the middle section from the other two sections and use it for
Stereo-seq Chip Slide mounting later.
Refer to Appendix I: Shipping Preservation and Transportation.

A paraffin section is divided into two sides, side A and side B. The side facing the
microtome is side A (matte side), and the side touching the blade is side B (smoothside). Always keep side B facing down when it is floating in the water bath, or keep it
mounted onto the Stereo-seq Chip Slide and microscopic glass slide to prevent thesection detachment.

Side ASide B

m. Trim the edges of the targeting section to fit the standard Stereo-seq Chip size (1 cm× 1 cm), and carefully transfer the section with side B facing down to the container
filled with 30% ethanol using a histology brush or clean forceps.

After trimming, the section should not exceed 0.9 cm x 0.9 cm because the tissue
section should not exceed 80% area coverage of the chip. An area exceeding 80% area
coverage of the chip will interfere with image and gene expression matrix registration,
resulting in misaligned results during bioinformatics analysis.

12

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manualn.

Use a clean microscopic glass slide to pick up the section and float it on the surfaceof the water in the preheated water bath. Make sure side B is facing down.

To prevent mix-ups when transferring adjacent sections, it is recommended thatyou transfer the adjacent sections to 30% ethanol and water bath separately andmount them onto pre-labeled microscopic glass slides in subsequent operations.

TissueSectionState

Possible Cause

Operation

The section
is completely
flattened without
folds.

Temperature isnormal/ideal

Proceed with theexperiment by following
the instructions.

Increase the water bathtemperature by 0.5°C
The section appears The temperature is~ 1°C, and observe
to be wrinkled
too low
continuously until thetissue is fully flattened.
Stop heating, add a small
The paraffin around
The temperature is amount of cold water, andthe section shows
too highthen scoop out the section
signs of melting
immediately.
o.

Wait until the tissue section is completely flattened in the water bath. Place theStereo-seq Chip Slide into the water bath and place the section near the chip. Usinga histology brush, pick up the section with the assistance of a histology brush by
gently touching the corner of the flattened section, ensuring that side B is in contact
with the Stereo-seq Chip.

13

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

Wipe off excess liquid from around and the back of the slide using dust-free paper
without touching the tissue, and then place it on the Stereo-seq PCR Adaptor in the
PCR thermal cycler. Dry the slide at 42°C for 3 hr and then bake it overnight at 37°C
according to the following incubation protocol.

p.

Temperature

Time(Heated lid) 45°C

on

42°C

∞

42°C

3 hr

37°C

∞

Stop Point:- After drying the tissue containing Stereo-seq Chip Slides on a PCR thermal cycler,
the slides can be stored in a slide container placed in an oven at 25°C or 37°C for
48 hr or at 4°C with desiccant for up to 1 week.

•

For detailed shipping guidance of paraffin-section-mounted Stereo-seq Chip Slides,
refer to Appendix I: Shipping Preservation and Transportation.

•

A technician with extensive experience in paraffin block sectioning can perform
section trimming and mounting according to their personal preferences.

•

Please pay attention to prevent air bubbles from forming when mounting thetissue section onto the chip surface.

•

The heating and baking process can also be substituted with a metal bath or aheating module that can be heated accurately at a constant rate.

14

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

Appendix I: Shipping Preservation and Transportation

Preservation and Transportation of FFPE Blocks
Store FFPE blocks at room temperature or at 4°C ~ 8 °C.
a.

Place the paraffin block in a sealed bag, then put the sealed bag into a foam box witha wall thickness of at least 3 cm, ensuring a tight seal.

b.

Use no fewer than 6 ice packs (13 cm × 23 cm) for a 24-hour shipment.

c.

Use foam, foam paper, and multiple layers of packaging to prevent sample damage
during transportation.

d. In summer, increase the number of ice packs to maintain sample temperatures

between 2°C ~ 8°C.

Preservation and Transportation of Paraffin-section-mounted Stereo-seq
Chip Slidesa.

After baking the tissue containing Stereo-seq Chip Slides at 42°C for 3 hr, transfer the
Stereo-seq Chip Slide into a slide container, and then place it in a sealable plasticbag. Place one desiccant pack per Stereo-seq Chip Slide into a sealable plastic bag,push out as much air as possible, and seal the bag tightly.

b.

Place the bag into a foam box with a wall thickness of at least 3 cm, ensuring a tight
seal.

c.

Use no fewer than 6 ice packs (13 cm × 23 cm) for a 24-hour shipment.

d. Use foam, foam paper, and multiple layers of packaging to prevent sample damage

during transportation.

e.

In summer, increase the number of ice packs to maintain sample temperatures
between 2°C ~ 8°C.

15

APPENDIX

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manual

When received, the paraffin-section-mounted Stereo-seq Chip Slides can be
stored in the original packaging at 4°C in dry conditions for up to 1 week.
f.

When retrieving the paraffin-section-mounted Stereo-seq Chip Slide from theshipping container or storage, take it out of the slide container and immediately
incubate it at 42°C with PCR Adaptor for 3 hr.

The temperature difference in the room during ice pack transportation may
cause water vapor to form on the chip's surface; therefore, it is important to dry
the chips as soon as possible.
g.

After drying the chips at 42°C, transfer the paraffin-section-mounted Stereo-seq Chip
Slide to a baking machine or a PCR thermal cycler and bake it at 60°C for 1 hr before
proceeding to the subsequent Transcriptomics experiments.

Preservation and Transportation of Paraffin Sectionsa.

Perform sectioning according to the instructions provided in Section 2.3. Choose theparaffin section that has the entire flat tissue surface revealed so that the section iscomplete and representative.
Sections that are incomplete, falling apart, or have obvious folds and wrinkles
are not acceptable for shipping and subsequent Transcriptomics experiments.
Obvious foldings and wrinkles

Incomplete section

16

Section exhibit completenessand smoothness

APPENDIX

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manualb.

Pick up the section with clean forceps or a histology brush and place it in a properly
labeled 50 mL centrifuge tube. Continue until all sections are collected. To prevent
sections from colliding during shipment and causing tears, no more than 5 sections
should be placed in one tube.

c.

Close the lid and place the pre-labeled centrifuge tube in a sealable bag with asufficient number of desiccants.

d. Place the bag into a foam box with a wall thickness of at least 3 cm, ensuring a tight

seal.

e.

e. Use no fewer than 6 ice packs (13 cm × 23 cm) for a 24-hour shipment.

f.

f. Use foam, foam paper, and multiple layers of packaging to prevent sample
damage during transportation.

g.

g. In summer, increase the number of ice packs to maintain sample temperatures
between 2°C ~ 8°C.

When received, the paraffin sections can be stored in the original packaging at 4°C
in dry conditions for up to 1 week.

17

APPENDIX

Stereo-seq Sample Preparation, Sectioning and Mounting Guide
for Formalin-fixed and Paraffin-embedded (FFPE) Samples on Stereo-seq Chip Slides User Manualh.

Static electricity may be generated during transportation, causing the sections
to adhere to the tube wall. Therefore, it is important to conduct a histology
examination before proceeding. When retrieving the sections, place the 50 mL
centrifuge tube at -20°C for 5 min, then pick up the section with clean forceps or ahistology brush.

i.

Flatten out the section in the water bath and then pick up the section with amicroscopic glass slide. Before proceeding to the subsequent experiments, performa routine H&E staining to ensure that the tissue has not been deformed.

j.

After the morphology of the tissue has been confirmed, follow the instructions
starting from Step h. in Section 2.3, Tissue Sectioning and Mounting, to float theparaffin section in 30% ethanol and then in a water bath.

k.

When the section is picked up and mounted on the Stereo-seq Chip Slide, proceedwith section drying and baking in Section 2.3, Tissue Sectioning and Mounting.

18

APPENDIX