Oct 31, 2024
An adaptable protocol for antibody & TDP-43 RNA aptamer dual immunohistochemical staining for FFPE-preserved human tissue: with SOP and tick-sheet.
- 1University of Aberdeen
External link: https://gregorylaboratory.com/protocols-sops/
Protocol Citation: Fergal M Waldron, Jenna Gregory 2024. An adaptable protocol for antibody & TDP-43 RNA aptamer dual immunohistochemical staining for FFPE-preserved human tissue: with SOP and tick-sheet.. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm97k6l3p/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: October 30, 2024
Last Modified: October 31, 2024
Protocol Integer ID: 111260
Keywords: Immunohistochemistry, TDP-43 RNA Aptamer , Antibody, Dual Staining, Amyotrophic Lateral Sclerosis, Frontotemporal Dementia, Alzheimer’s Disease, Pathology
Funders Acknowledgement:
Target ALS
Grant ID: BB-2022-C4-L2
Abstract
Here we provide a protocol with SOP and tick-sheet, for correct procedures to dual stain FFPE human tissue for, 1) pathological TDP-43 protein, along with 2) any other protein of interest, with the aim of visualising protein of interest histology in the context of TDP-43 pathology.
This method utilises a published TDP-43 RNA aptamer (biotinylated)(Spence and Waldron, 2024 in Acta Neuropathologica here https://link.springer.com/article/10.1007/s00401-024-02705-1) which detects pathological TDP-43 with greater sensitively and specificity than with current antibody approaches.
This protocol can be implemented for dual staining of any rabbit or mouse primary detection antibody (using the Novolink Polymer Detection System), and with our TDP-43 RNA aptamer (Spence and Waldron, 2024)
The resulting dual stain will show brown chromogen (detecting the target of the primary antibody) with red chromogen(detecting TDP-43 pathology with our TDP-43 RNA aptamer).
Reference for citation of this method
To cite this dual staining method, please cite Waldron and Gregory, 2024 (here) published on protocols.io:
An adaptable protocol for antibody & TDP-43 RNA aptamer dual immunohistochemical staining for
FFPE-preserved human tissue: A SOP and tick-sheet.
Fergal M. Waldron‡, Jenna M. Gregory‡, (2024). Protocols.io 2024;
DOI: dx.doi.org/10.17504/protocols.io.yxmvm97k6l3p/v1
‡equal contributions and co-corresponding authors
Image Attribution
Jenna Gregory
Guidelines
For users attention
Sequenza immunostaining racks: This protocol is designed for use with Sequenza immunostaining racks
Essential fixation step (end of day 2): The TDP-43 RNA aptamer published in Zacco et al., 2022 was designed for flexible use, with moderate binding affinity (e.g. as desirable for some single molecule imaging applications). To improve TDP-43 RNA aptamer binding for staining human tissue, Spence and Waldron et al., 2024 employ an overnight fixation step after incubation with TDP-43 RNA aptamer. This fixation step is
essential for reliable and reproducible tissue staining with TDP-43 RNA aptamer. Without the fixation step, the aptamer will not bind with a high enough affinity resulting in partial staining with potential to lead to misleading and erroneous pathological conclusions.
Steric hindrance occurs when molecules (here, antibody and aptamer) affect their respective binding to their target antigens, with the potential for reduced or absence of detectable fluorescence signals.
Therefore , it is imperative that users first optimise single staining protocols for both their primary antibody (mouse or rabbit only for compatibility with the Novolink Polymer Detection System), and for TDP-43Apt, before attempting the dual stain outlined here.
Before start
See Appendix A for materials, Appendix B solution recipes, and Appendix C for pressure cooker instructions.
Materials
Biological materials
• FFPE tissue slides
Reagents
Histology
• Xylene
• Ethanol
• Haematoxylin
• Lithium carbonate
• DPX mountant (or similar)
Immunohistochemistry
• Citric acid
- 5N hydrochloric acid & 5N sodium hydroxide to bring citric acid to pH 6
• MilliQ H2O
• Distilled H20
• Novolink Polymer Detection System Kit (Leica Biosystems; ???)
• Peroxidase block (3% H2O2) – included in Novolink Polymer Detection Kit
• TBS
• Avidin/Biotin Block: e.g. Avidin/Biotin Blocking Kit (Abcam, ab64212)
• TDP-43 Aptamer (TDP-43APT): The sequence is: CGGUGUUGCU with a 3' Biotin-TEG modification, purified using HPLC, scale: 1.0 µM synthesis (as published in Nature Communications here doi: 10.1038/s41467-022-30944-x by Zacco et al., 2022).
• 4% w/v Formaldehyde: from e.g. Pierce‱ 16% Formaldehyde (w/v), Methanol-free (ThermoFisher Scientific, Cat No. 28906
• Anti-Biotin−Alkaline Phosphatase antibody produced in goat (Abcam; ab6652)
• Fast Red Chromogen (available from Leica BioSystems)
Equipment
Histology
• Fume hood (for use with Xylene)
• Slides and coverslips
• Histology staining racks and pots
• Coplan jars or similar
Immunohistochemistry
• pH meter (to make citric acid up to pH 6)
• Sequenza rack and coverplates
• Pressure cooker (for antigen retrieval): e.g. Drew and Cole Pressure King Pro
• Refrigerator
• Plastics and other (Pipettes and tips, Falcon tube, dropper, eppendorfs)
Safety warnings
Safety First
Before starting, please seek out and read all relevant Health & Safety documentation, and fully read this SOP.
Before working please make sure these have been carried out.
> COSHH assessment
> Risk assessment
> Safe System of Work
> SOP read and understood
Before start
See Appendix A for materials, Appendix B solution recipes, and Appendix C for pressure cooker instructions.
Protocol references
Reference for citation of this method
To cite this dual staining method, please cite Waldron and Gregory, 2024 (here) published on protocols.io:
An adaptable protocol for antibody & TDP-43 RNA aptamer dual immunohistochemical staining for
FFPE-preserved human tissue: A SOP and tick-sheet.
Fergal M. Waldron*, Jenna M. Gregory* (2024). Protocols.io 2024;
DOI: dx.doi.org/10.17504/protocols.io.yxmvm97k6l3p/v1
*equal contributions and co-corresponding authors
Other relevant publications
The citation, Spence and Waldron et al., 2024, for detection of pathological TDP-43 in human tissue using the TDP-43 RNA aptamer (TDP-43APT) published in Acta Neuropathologica:
RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS.
Holly Spence*, Fergal M. Waldron*, Rebecca S. Saleeb, Anna-Leigh Brown, Olivia M. Rifai, Martina Gilodi, Fiona Read, Kristine Roberts, Gillian Milne, Debbie Wilkinson, Judi O’Shaughnessy, Annalisa Pastore, Pietro Fratta, Neil Shneider, Gian Gaetano Tartaglia, Elsa Zacco, Mathew H. Horrocks, Jenna M. Gregory‡ (2024). Acta Neuropathologica 2024 Mar 5;147(1):50. DOI: 10.1007/s00401-024-02705-1
*equal contributions, ‡corresponding author
The citation, Zacco et al., 2022, for design of the TDP-43 RNA aptamer (TDP-43APT) to visualize TDP-43 condensates with super-resolution microscopy published in Nature Communications:
Probing TDP-43 condensation using an in silico designed aptamer.
Elsa Zacco, Owen Kantelberg, Edoardo Milanetti, Alexandros Armaos, Francesco Paolo Panei, Jenna M. Gregory, Kiani Jeacock, David J. Clarke, Siddharthan Chandran, Giancarlo Ruocco, Stefano Gustincich, Mathew H Horrocks, Annalisa Pastore, Gian Gaetano Tartaglia (2022). Nature Communication 2022 Jun
23;13(1):3306. doi: 10.1038/s41467-022-30944-x.
The citation, Rifai et al., 2024, for the first publication employing dual staining for NEK1 and TDP-43 in Brain
Pathology:
Clinicopathological analysis of NEK1 variants in amyotrophic lateral sclerosis.
Olivia M. Rifai, Fergal M. Waldron, Danah Sleibi, Judi O’Shaughnessy, Danielle J. Leighton*, Jenna M. Gregory*‡ (2024). Brain Pathology DOI: 10.1111/bpa.13287
*equalcontributions, ‡corresponding author
Acknowledgements
The authors thanks Target ALS for funding this work (Grant ID: BB-2022-C4-L2).