Sep 18, 2025
FFPE Human Brain Tissue Quality Control for Xenium
- Tsering Lama1,
- Wenqing Cao1,
- Xiaoying(Miya) Lai1,
- Philip De Jager1,
- Mariko Taga1,
- YA ZHANG1
- 1The Center for Translational & Computational Neuroimmunology, Department of Neurology, Columbia Univerisity Neuroimmunology Core Neuroimmunology Core Columbia University Medical Center
- CUIMC
Protocol Citation: Tsering Lama, Wenqing Cao, Xiaoying(Miya) Lai, Philip De Jager, Mariko Taga, YA ZHANG 2025. FFPE Human Brain Tissue Quality Control for Xenium. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6z841gqe/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: September 04, 2025
Last Modified: September 18, 2025
Protocol Integer ID: 226396
Keywords: Xenium, FFPE, FFPE Human Brain Tissue, DAPI Staining, H&E Staining, histology, Spatial Transcriptomic, immunohistochemistry, ffpe human brain tissue quality control for xenium, xenium in situ gene expression platform, ffpe human brain tissue quality control, selection workflow for ffpe human brain tissue, ffpe human brain tissue, xenium assay, integrity of ffpe tissue block, quality xenium analysis, ffpe tissue block, human brain tissue, xenium, 10x genomic, situ gene expression platform, targeted protein marker, molecular quality check, protein marker, subcellular resolution, tissue quality, molecular profiling, rna detection at subcellular resolution, brain region
Abstract
The Xenium In Situ Gene Expression platform from 10x Genomics enables high-plex, targeted RNA detection at subcellular resolution, supporting spatially resolved cell typing and molecular profiling. While an optimized workflow has been provided by 10x, formalin-fixed paraffin-embedded (FFPE) post-mortem human brain tissue presents unique challenges due to its heterogeneity and lipid-rich composition. In particular, the quality and integrity of FFPE tissue blocks vary considerably across donors and brain regions. Rigorous quality control prior to Xenium assays is necessary.
In this protocol, we describe a standardized pre-assay QC and region of interest (ROI) selection workflow for FFPE human brain tissue. Tissue sections undergo deparaffinization followed by histological and molecular quality checks, including hematoxylin and eosin (H&E) staining to assess morphology, DAPI staining to evaluate nuclear integrity, and immunofluorescent staining to identify ROI regions. These QC steps enable the identification of regions with preserved morphology and targeted protein markers, ensuring that selected ROIs are biologically relevant and suitable for high-quality Xenium analysis.
Guidelines
This protocol is for quality check of FFPE tissue blocks and selection of regions of interest (ROIs) for Xenium experiments.
Materials
1. CitriSolv (d-limonene) Clearing Agent – Decon Laboratories, Inc.
CN: 1601H
2. 100% Ethanol – Fisher Chemical
CN: A4094
3. 70% Ethanol – Decon Laboratories, Inc.
CN: 2405
4. Citrate Buffer pH 6 – Sigma-Aldrich
CN: C9999
5. 10X Phosphate-Buffered Saline – Quality Biological
CN: 119-069-131
6. Bovine Serum Albumin – Sigma-Aldrich
CN: A7906
7. TrueBlack Lipofuscin Autofluorescence Quencher – Biotium
CN: 23007
8. ProLong Gold DAPI Mountant – Invitrogen
CN: P36931
9. ProLong Glass Mountant – Invitrogen
CN: P36984
10. Cover Glass – ThorLabs Inc.
CN: CG15KH
11. Glass Slides – Globe Scientific Inc.
CN: 1358W
12. Harris Hematoxylin Solution – Sigma-Aldrich
CN: HHS32
13. Eosin Solution – Sigma-Aldrich
CN: 1098442500
14. Shandon Bluing Reagent – Epridia
CN: 6769001
15. Microscope Slides – Globe Scientific
CN: 1358W
Troubleshooting
Safety warnings
N/A
Ethics statement
All brain specimens were derived from two longitudinal clinico-pathological cohort studies, that is, the ROS and MAP. In both cohorts, participants did not have known dementia at the time of enrollment. Participants agreed to receive clinical evaluation each year and to donate their brain at the time of death. Each study was approved by the institutional review board of Rush University Medical Center. All participants signed a written informed consent, Anatomical Gift Act and repository consent.
Before start
Prior to the quality check steps in this protocol, prepare a microscope slide mounted with an intact tissue section from your interested block. Refer to the Block Facing, Rehydration and Sectioning sections from "FFPE Human Brain Tissue Serial Sectioning for Xenium" protocol for specific procedures.
Tissue Deparaffinization
Incubate the tissue sections at 60°C for 30 min
Cool down the slides at room temperature (RT) for 7 min
Incubate the tissue sections in Xylene for 10 min. Repeat once
Incubate the tissue sections in 100% Ethanol for 3 min. Repeat once
Incubate the tissue sections in 96% Ethanol for 3 min. Repeat once
Incubate the tissue sections in 70% Ethanol for 3 min
Wash the tissue sections with Milli-Q water for 20 sec. Immediately proceed to H&E staining.
H&E Staining
Incubate the tissue sections in Milli-Q water for 2 min
Incubate the tissue sections in Hematoxylin solution for 20 min
Wash the tissue sections with Milli-Q water for 1 min. Repeat twice
Incubate the tissue sections in Bluing Solution for 2 min
Wash tissue with Milli-Q water for 2 min
Incubate the tissue sections with 70% Ethanol for 3 min
Incubate the tissue sections with 95% Ethanol for 3 min
Incubate the tissue sections with Eosin Solution (400:1 mixture of Eosin and 0.1M Hydrochloric acid) for 3 min
Incubate the tissue sections in 95% Ethanol for 30 sec. Repeat once
Incubate the tissue sections in 100% Ethanol for 30 sec. Repeat once
Incubate the tissue sections in Xylene for 3 min. Repeat once
Let the tissue sections dry completely. Mount the tissue slide by adding ~100 uL of Prolong Glass, depending on tissue size, on the tissue section.
Coverslip tissue slide by taking a cover glass and placing one finger on one side of the cover
glass. Then, slowly and gently lower the cover glass using your other hand to guide the coverglass down until the entire slide is coverslipped. Avoid bubbles.
Cover glass placement
H&E staining of FFPE human brain tissue at 20X.
Note
There are two suggested options for performing the incubation steps:
- A glass staining jar or 50 mL conical tube can be used to insert slides and submerge tissue in the solution.
- Another option is adding approximately 250 uL (may vary depending on tissue size) of solution directly on the tissue ensuring the entire tissue section is covered. This option requires more thorough washing using a wide mouth wash bottle.
DAPI and Immunofluorescent Staining for Region of Interest (ROI) Selection
Deparaffinize the tissue sections with a clearing agent for 20 min. This protocol uses d-limonene as a non-toxic Xylene alternative.
Immediately rehydrate tissue with graded ethanol washes starting with 100% Ethanol twice
and then 70% Ethanol
Wash the tissue slide with 1X phosphate-buffered saline (PBS) for 3 times at RT
Perform heat-induced antigen retrieval using citrate buffer in a microwave (800W, 30% power) for 25 min
Wash the tissue sections with 1X PBS for 3 times at RT
Add 3% bovine serum albumin (BSA) for 30 min as a blocking agent atRT
Incubate the tissue sections with primary antibodies diluted in 1% BSA with 1X PBS overnight at 4°C
Note
The antibodies used in the immunofluorescent staining will depend on the project’s objectives.
Wash the tissue sections slide with 1X PBS for 3 times at RT
Incubate the tissue sections with Alexa Fluor secondary antibodies diluted in 1% BSA with 1X PBS for 1 hr at RT
Wash the tissue sections with 1X PBS for 3 times at RT
Treat the tissue sections with 1X TrueBlack lipofuscin quencher for 2 min at RT to minimize autofluorescence
Wash the tissue sections with 1X PBS for 3 times at RT
Mount slide by adding ~100 uL of an anti-fading reagent containing DAPI to visualize nuclei
Coverslip slide by slowly and gently releasing the cover glass over the tissue slide
Note
When coverslipping, ensure the tissue is not too dry to prevent air bubbles and to allow proper adhesion of the cover glass to the tissue slide. If air bubbles form, use a 10 uL pipette tip on top of the cover glass to gently guide the bubbles away from the tissue toward the edge of the slide.
Visualize the staining using a fluorescent microscope to determine an appropriately sized tissue area for ROI selection
DAPI staining of FFPE human brain tissue at 20X.
Note
The ROI selected for this protocol is approximately 8 mm × 6 mm in size to accommodate three sections within the capture area. ROIs will be selected according to the experiment objectives.
4X Brightfield image of whole tissue section with ROI
annotated in yellow box