**Biomaterials**

A human lung cancer tissue block (FFPE) was obtained from Avaden Biosciences.

- Sex: Female
- Age: 72 years

**Sample preparation**

A 5 µm section was taken from the FFPE tissue block with a microtome (Epredia HM355S). Sectioning, deparaffinization, staining and imaging followed the Visium HD FFPE Tissue Preparation Handbook ([CG000684](/support/spatial-gene-expression-hd/documentation/steps/tissue-prep-for-ffpe/visium-hd-ffpe-tissue-preparation-handbook)).

Antibodies used:

- Channel 1 - Alexa Fluor 594 anti-CD45RO (clone UCHL1, Biolegend 304254, dilution 1:100)
- Channel 2 - Alexa 488 anti-CD20 (clone EPR459Y; Abcam ab198941, dilution 1:100)
- Channel 3 - DAPI

**Imaging**

- Image type: IF
- Microscope: Olympus VS200 Slide Scanner
- Objective magnification: 20X
- Numerical Aperture: 0.8
- ScopeLED light source: Excelitas X-Cite NOVEM-L
- Camera: Hamamatsu Orca FusionBT sCMOS (2304 x 2304 pixels (16bits), 6.5um pixel size, 95% QE, 89.1 fr/sec, ~1Gb/sec)
- Exposure: DAPI (ex 385 nm, em 447 nm): 0.75 microseconds; 488 (ex 466 nm, em 515 nm): 125 microseconds; 590 (ex 586 nm, em 628 nm): 50 microseconds

**Assay workflow**

Probe hybridization, probe ligation, slide preparation, probe release, extension, and library construction followed the Visium HD Spatial Gene Expression Reagent Kits User Guide ([CG000685](/support/spatial-gene-expression-hd/documentation/steps/library-construction/visium-hd-spatial-gene-expression-reagent-kits-user-guide)).

- Slide serial number: H1-HMD7CWH
- Area: A-1
- Instrument: Visium CytAssist
- Probe set: Visium Human Transcriptome Probe Set v2.0

**Sequencing**

- Indexing: Dual index plate TS set A; sample index C2
- Sequencing instrument: Illumina NovaSeq 6000
- Sequencing configuration: 43 bp read 1, 50 bp read 2, 10 bp i7 sample index, and 10 bp i5 sample index
- Sequencing depth: 357.6 million reads

**Analysis**

Space Ranger v3.0 was used to map FASTQ files to the reference, detect tissue, align the data to the microscope and CytAssist images, and output feature-barcode matrices for further analysis.

**How to view data**

To get started, [download Loupe Browser v8.0](/support/software/loupe-browser/downloads) to explore the Loupe file, or read more about the other [Space Ranger outputs](/support/software/space-ranger/latest/analysis/outputs/output-overview). We recommend that users turn on "auto-image adjustment" when exploring the data in Loupe in order to improve visibility of the IF image.


CytAssist image

Microscope image

Loupe alignment file

FASTQs

Probe Set CSV

Slide layout file

Web Summary HTML

Loupe file

Feature slice H5

Metrics summary CSV

Molecule info H5

Spatial outputs

Binned outputs (all bin levels)

Web Summary

Files

Visium CytAssist

Visium

Gene Expression

Protein co-detection

Compact instrument designed to simplify the Visium workflow by facilitating the transfer of analytes from standard glass slides to Visium slides.

HD Spatial Gene Expression

Whole transcriptome probe-based gene expression assay at single cell scale, compatible with FFPE, Fresh Frozen, and Fixed Frozen tissue sections

Space Ranger

Visium Spatial Gene Expression is a next-generation molecular profiling solution for classifying tissue based on total mRNA. Map the whole transcriptome with morphological context in FFPE or fresh frozen tissues to discover novel insights into normal development, disease pathology, and clinical translational research.

Immunofluorescence protein co-detection

Visium CytAssist Gene and Protein Expression

Map the whole transcriptome within the <em>tissue context</em>

Spatial Gene Expression

Spatial gene expression profiling and imaging data of fresh frozen or FFPE tissues.

Analyze your Visium Spatial data with microscope images.

Visium HD Spatial Gene Expression Library, Human Lung Cancer, IF Stained (FFPE)

<SupportCallout level={"info"}>This assay requires CytAssist instrument Firmware v2.0.0 or higher. Refer to the [Visium CytAssist with Readiness Test User Guide (CG000542)](/support/instruments/visium-cytassist/visium-cyt-assist-with-readiness-test) for details.</SupportCallout>
<br />

<Arcade
id="key-techniques-visium-hd-slide-preparation"
label={"Best Practice video series"}
title={"Key Techniques for Visium HD Slide Preparation & Use"}
arcadeId={"wi3xFe9UVxkTpz8JIUyf"}
/>

<Arcade
id="tissue-slide-handling-and-use"
label={"Best Practice video series"}
title={"Key Techniques for Tissue Slide Handling & Use"}
arcadeId={"RFM1pJNdetKs3zfc4GFk"}
/>

This document outlines the protocol for generating Visium HD Spatial Gene Expression libraries from FFPE, Fresh Frozen, and Fixed Frozen tissue sections. Key companion documents for use with this document:

1. Visium HD Spatial Gene Expression Protocol Planner (CG000698)
2. Tissue Slide Alignment Instruction, Quick Reference Cards (CG000548)
3. Visium Cassette S3 for Assembly and Disassembly Quick Reference Cards (CG000730)

For use with:

- Visium HD, Human Transcriptome, 6.5 mm, PN-1000675 (4 rxns) / PN-1000673 (16 rxns)
- Visium HD, Mouse Transcriptome, 6.5 mm, PN -1000676 (4 rxns) / PN-1000674 (16 rxns)
- Visium CytAssist Reagent Accessory Kit, PN-1000499
- Dual Index Kit TS Set A, 96 rxns, PN-1000251


This document outlines the protocol for generating Visium HD Spatial Gene Expression libraries from FFPE, Fresh Frozen, and Fixed Frozen tissue sections.

Visium HD Spatial Gene Expression Library, Human Lung Cancer, IF Stained (FFPE)

Assess data quality

Visualize and explore data