Dec 11, 2025
Visium sample preparation from human Midbrain
- Nathan Haywood1,2,
- Geidy Serrano3,2,
- Madison Cline3,2,
- Zhixiang Liao4,2,
- Idil Tuncali4,2,
- Thomas G Beach3,2,
- Joshua Levin5,2,
- Clemens R Scherzer2,6,7,8,9
- 1Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
- 3Banner Sun Health Research, Sun City, AZ 85351, USA;
- 4Brigham and Women's Hospital;
- 5Broad Institute of MIT and Harvard;
- 6Stephen & Denise Adams Center for Parkinson’s Disease Research of Yale School of Medicine, New Haven, CT 06510;
- 7APDA Center for Parkinson Precision Medicine, Yale, New Haven, CT 06510;
- 8Department of Neurology, Yale, New Haven, CT 06510;
- 9Department of Genetics, Yale, New Haven, CT 06510
- Scherzer Lab
Protocol Citation: Nathan Haywood, Geidy Serrano, Madison Cline, Zhixiang Liao, Idil Tuncali, Thomas G Beach, Joshua Levin, Clemens R Scherzer 2025. Visium sample preparation from human Midbrain. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpwdkvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 25, 2025
Last Modified: December 11, 2025
Protocol Integer ID: 233490
Keywords: ASAPCRN, run visium spatial transcriptomics samples from human midbrain, run visium spatial transcriptomics sample, visium sample preparation from human midbrain, visium sample preparation, human midbrain
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000301
Abstract
This protocol describes a workflow to prepare and Run Visium Spatial Transcriptomics samples from human Midbrain.
Brain sample sectioning and placement
Single 10µm brain sections were prepared following strictly the Visium Spatial Protocol - Tissue Preparation guide by 10x genomics (CG000240 | Rev C, 10x Genomics) and placed onto Visium spatial gene expression slides ( #PN-1000184, 10x Genomics).
Per subject two 10µM sections were prepared to cover both the ventral and dorsal part of the midbrain (see image for ref) and placed on a visium spatial slide.
Visium spatial slides were shipped overnight collaborator on dry ice and stored at -80 °C for no more than a week before processing was continued.
Methanol Fixation H&H staining | QC Imaging
Visium slides were fixed with Methanol and subjected to H&E Staining following 10x Genomics Methanol Fixation, H&E Staining & Imaging for Visium Spatial Protocols (CG000160 | Rev B, 10x Genomics) before imaging.
Slides were imaged on an Axio Imager.Z2 microscope (Zeiss) with Metafer 5 (v3.14.150, RRID:SCR_016306 MetaSystems) automated software at 10X magnification, 1.0 CCD camera gain, and fixed lamp RGB levels.
Visium spatial gene expression library preparation & Sequencing parameters
Samples were processed as directed following strictly Visium spatial gene expression Reagent kits user guide (CG000239 |Rev D, 10x Genomics) using the Visium spatial Gene Expression Reagent Kit (#PN-1000186, 10xGenomics) and the Library Construction Kit (#PN-1000190, 10xGenomics). The permeabilization time was optimized as directed (CG000238 | Rev D, 10x Genomics).
Tissue optimization experiments were imaged using identical Metafer 5 settings following the H&E staining as described in step 4.
Fluorescence imaging was performed with an Agilent Technologies DNA MicroArray Scanner and analyzed using Agilent Feature Extraction (v10.5.1.1, RRID_014963, Agilent Technologies) software.
Libraries were pooled for each section, and sequenced to a depth of at least ~50,000 reads per spot on an Illumina NovaSeq with an SP flow cell with read lengths of 28 (Read 1), 90 (Read 2), 10 (Index 5), and 10 bases (Index 7).
Protocol references