Feb 25, 2026
FFPE Post-Xenium Iterative Indirect Immunofluorescence Imaging (4i)
- Tsering Lama1,
- Berke Karaahmet1,
- Philip De Jager1,
- Mariko Taga1
- 1The Center for Translational & Computational Neuroimmunology, Department of Neurology, Columbia University Medical Center
- Neuroimmunology core
Protocol Citation: Tsering Lama, Berke Karaahmet, Philip De Jager, Mariko Taga 2026. FFPE Post-Xenium Iterative Indirect Immunofluorescence Imaging (4i) . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2zpyql1y/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: February 24, 2026
Last Modified: February 25, 2026
Protocol Integer ID: 243967
Keywords: FFPE, Human brain tissue, FFPE human brain tissue, Spatial Transcriptomics, Xenium, 10 genomics, Immunofluorescence, Immunohistochemistry, Iterative Indirect Immunofluorescence Imaging, 4i, whole-tissue imaging, imaging, scanning, post-Xenium, post Xenium, protein, morphology, tissue architecture, tissue organization, pathology, neurodegeneration, multiplex, staining, IHC, IF, antibody elution, spatial transcriptomic, gene expression profiling at subcellular resolution, aligning spatial transcriptomic data, iterative indirect immunofluorescence imaging, spatial transcriptomic data with proteomic information, situ gene expression platform, 10x genomic, subcellular resolution, 4i workflow for xenium, ffpe tissue, use of ffpe tissue, human brain tissue, xenium processing, gene expression profiling, pathological protein marker, preserving tissue architecture, following xenium processing, neuropathological marker, tissue architecture, powerful insights into tissue organization, rna localization, 4i workflow
Abstract
Spatial transcriptomics (ST) enables high-plex, in situ gene expression profiling at subcellular resolution while preserving tissue architecture, offering powerful insights into tissue organization. However, current ST workflows are limited in resolving morphologically complex cell types when relying solely on RNA localization. Additionally, ST platforms do not directly capture pathological protein markers. To address these limitations, we developed an integrated post-spatial profiling workflow that combines the XeniumTM In Situ Gene Expression platform from 10x Genomics with iterative indirect immunofluorescence imaging (4i) on formalin-fixed paraffin-embedded (FFPE) post-mortem human brain tissue. This approach enables multiplexed, multi-omic mapping of the pathological microenvironment from the same tissue section, aligning spatial transcriptomic data with proteomic information. The use of FFPE tissue best preserves cellular morphology following Xenium processing and expands the range of markers available for multiplex staining. By incorporating cell-type-specific and neuropathological markers into a customizable 4i panel, this workflow improves transcript-to-cell assignment, provides spatially resolved insight into disease-associated cellular changes and interactions, and is adaptable for diverse approaches to downstream analyses.
In this protocol, we describe a 4i workflow for Xenium processed 5 μm FFPE human brain tissue sections, that can generate up to a 20-plex image. We outline key procedures for post-Xenium sample processing, antigen retrieval, recommended tissue washing to prevent microtears, and an optimized β-mercaptoethanol (βME)-based stripping solution for efficient antibody elution.
Additionally, we recommend following our related Xenium protocols from our Neuroimmunology Core profile, which provide best practices for tissue quality control and serial sectioning to ensure high quality staining performance post-processing.
Guidelines
This protocol details an optimized workflow for 4i post-Xenium on FFPE human brain tissue, highlighting important sample processing tips, antigen retrieval recommendations, and an effective stripping solution for antibody elution.
Materials
βME Stripping Solution
SDS 10% Solution cat#AM9822 (Invitrogen)
1 M Tris-HCl pH 6.8 cat#ab286853 (Abcam)
2-Mercaptoethanol cat#63689 (Sigma-Aldrich)
TBS-Tween 20X Buffer cat#28360 (Thermo Scientific)
Glass staining jar cat#900620 (DWK Life Sciences)
IF
Slide Mailer cat#HS15986 (Fisherbrand)
Slide Tray cat# 195801 (Research Products International Corp)
Citrate Buffer pH 6 cat#C9999 (Sigma-Aldrich)
10X PBS cat#119-069-131 (Quality Biological)
Normal Donkey Serum cat#017-000-121 (Jackson ImmunoResearch)
True Black cat#23007 (Biotium)
ProLong Gold DAPI Mountant cat#P36931 (Invitrogen)
ProLong Glass Mountant cat#P36984 (Invitrogen)
Cover Glasses cat#CG15KH (ThorLabs Inc.)
Glass Slides cat#1358W (Globe Scientific Inc.)
Troubleshooting
Problem
Microtears on tissue sections may occur due to repeated washing and coverslip removal.
Solution
To minimize microtears during the 4i assay, perform washes using a slide mailer to allow gentler but thorough washing compared to traditional methods like a wide-mouth wash bottle. For coverslip removal, soften the mounting medium by submerging the tissue slide in water prior to removal. When using an edge blade, we recommend beginning at one corner of the glass coverslip and gently lifting upward, especially closer to the tissue section. Controlled speed and force are critical for proper coverslip removal to prevent tissue damage.
Problem
High background signal in immunofluorescence staining may occur if tissue slides are stored in PBS-T for longer than one week following Xenium processing or if washing is insufficient. Extended storage of post-Xenium tissue slides in PBS-T without buffer changes can lead to tissue degradation or contamination, resulting in non-specific antibody binding.
Solution
Samples must be processed for immunofluorescence staining within one week of Xenium processing.
Safety warnings
Please follow proper chemical safety and disposal guidelines when handling βME and sharps. Always wear the appropriate personal protective equipment when performing this experiment.
Ethics statement
This study was approved by the Institutional Review Board of Columbia University (AAAR4962). All human tissue donors provided informed consent, permitting the use of their postmortem brains and associated antemortem clinical data for research purposes.
Before start
Post-Xenium Sample Processing
Iterative indirect immunofluorescence (4i) staining was performed on 5 μm FFPE human brain tissue sections following Xenium processing. Post-Xenium tissue slides can be stored in 1X phosphate-buffered saline containing Tween-20 (PBS-T) at 4°C for up to 7 days, with recommended buffer changes every 2-3 days. Tissue sections were washed with 1X PBS-T prior to immunofluorescence staining.
Immunofluorescence Staining
Retrieve Xenium slides stored in slide mailer with 1X PBS-T
Gently wash tissue with 1X PBS-T X3
Note
We recommend using end-opening slide mailers to allow gentle, efficient washing and to prevent microtears or tissue detachment from the slide.
Prepare 400 mL of 1X Citrate pH 6 antigen retrieval in Milli-Q H2O
Note
We prepare 360 mL of Milli-Q H2O in a clean 500 mL graduated cylinder and add up to 400 mL of 10X Citrate pH 6.
Microwave tissue in antigen retrieval at 400 W (30% power) for 25 min
Wash tissue with 1X PBS-T X3
Incubate tissue with 88% Formic Acid for 2 min (if applicable)
Note
When using Aβ markers, we recommend treating tissue with 88% formic acid and washing the slide with Milli-Q H2O.
Wash tissue with 1X PBS-T X3
Block tissue with 5% Normal Donkey Serum (NDS) for 30 min
Incubate tissue with primary antibodies in 1% NDS prepared in 1X PBS at 4°C overnight
Wash tissue with 1X PBS-T X3
Incubate tissue with secondary antibodies in 1% NDS/PBS for 1 hour
Wash tissue with 1X PBS-T X3
Block tissue with 5% NDS for 30 min (if applicable)
Note
We recommend blocking the tissue if a conjugated antibody is used in the staining cycle.
Incubate tissue with conjugated antibodies in 1% NDS/PBS for 1 hour (if applicable)
Incubate tissue with 1X TrueBlack for 2 min
Note
1X TrueBlack was prepared by combining 70% ethanol and 20X TrueBlack. The solution can be stored long-term in a light-sensitive tube at room temperature (RT).
Wash tissue with 1X PBS-T X3
Mount tissue with Antifade ProLong Gold DAPI
Coverslip slide and let dry overnight @ RT (long-term storage @ 4°C in slide box)
Perform image acquisition to prepare for next round of staining
Preparing the βME Stripping Solution (100 mL recipe)
Prepare a sterile media bottle for βME stripping solution
Add 20 mL of 10% Sodium Dodecyl Sulfate (SDS)
Add 6.25 mL of 1M Tris-HCl pH 6.8
Microwave 400 mL of Milli-Q H2O at 100% power for 2 min
Note
The water temperature should be about 60°C.
Add up to 100 mL of microwaved Milli-Q H2O to media bottle
To confirm the stripping solution has a pH value of 6.8, use a pH meter and adjust as needed
Under the fume hood, add 800 μL of βME to the solution
Note
When working with βME, ensure proper personal protective equipment (PPE) is worn and safe handling is performed based on institutional guidelines.
βME Stripping Process
Turn on incubator and set to 60°C
Gently remove coverslip from slide
Note
We recommend using a sterile edge blade to carefully remove coverslip. When handling sharps, ensure proper PPE and safe disposal.
Wash tissue with 1X PBS-T X3
Incubate tissue in βME stripping solution at 60°C for 45 min
Note
We recommend using glass staining jars for stripping incubation.
Wash tissue with Milli-Q H2O on shaker for 1 hour with water changes every 15 minutes
Wash tissue with 1X PBS-T X3
Incubate tissue with 1X TBS-T for 5 min
Wash tissue with 1X PBS-T X3
Incubate tissue with 1X TrueBlack for 5 min
Wash tissue with 1X PBS-T X3
Mount tissue with ProLong Glass
Coverslip slide
Confirm proper antibody elution under the microscope
Gently remove coverslip from slide
Wash tissue sections with 1X PBS-T X3
Block tissue with 5% NDS for 30 min
Start new round of staining
Protocol references
10x Genomics. Xenium in situ spatial profiling for FFPE – tissue preparation guide. CG000578, Rev E. Pleasanton (CA): 10x Genomics 2025. Available from: https://www.10xgenomics.com/support/in-situ-gene-expression/documentation/steps/tissue-prep-ffpe/xenium-in-situ-spatial-profiling-for-ffpe-%E2%80%93-tissue-preparation-guide
Cao W, Lama T, Lai X(Miya), De Jager P, Taga M, Zhang YA. FFPE human brain tissue serial sectioning for Xenium. protocols.io 2025. doi:10.17504/protocols.io.6qpvrwk9blmk/v1.
Gendusa R, Scalia CR, Buscone S, Cattoretti G. Elution of High-affinity (>10-9 KD) Antibodies from Tissue Sections: Clues to the Molecular Mechanism and Use in Sequential Immunostaining. J Histochem Cytochem. 2014 Jul;62(7):519-31. doi: 10.1369/0022155414536732. Epub 2014 May 2. PMID: 24794148; PMCID: PMC4174624.
Lama T, Cao W, Lai X(Miya), De Jager P, Taga M, Zhang YA. FFPE human brain tissue quality control for Xenium. protocols.io 2025. doi:10.17504/protocols.io.bp2l6z841gqe/v1.