Aug 28, 2024

Public workspaceAutomated immunodetection of proteinase-K-resistant Lewy Pathology in FFPE human colon tissue V.1

DOI
dx.doi.org/10.17504/protocols.io.kxygxy8ekl8j/v1
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Elizabeth Videlock
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DOI: dx.doi.org/10.17504/protocols.io.kxygxy8ekl8j/v1
Protocol Citation: Elizabeth Videlock 2024. Automated immunodetection of proteinase-K-resistant Lewy Pathology in FFPE human colon tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxy8ekl8j/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: August 28, 2024
Last Modified: August 28, 2024
Protocol Integer ID: 106608
Funders Acknowledgement:
Chan Zuckerberg Initiative
Grant ID: 2021-235154
Abstract
Automated detection of gut Lewy Pathology using Leica Bond RX processor based on Protocol F using Bond Polymer Refine Detection kit (Leica Biosystems, Cat#: DS9800). Adapted from "Method 5" as described in Beach TG et al., J Parkinsons Dis. 2016.
CITATION
Beach TG, Corbillé AG, Letournel F, Kordower JH, Kremer T, Munoz DG, Intorcia A, Hentz J, Adler CH, Sue LI, Walker J, Serrano G, Derkinderen P (2016). Multicenter Assessment of Immunohistochemical Methods for Pathological Alpha-Synuclein in Sigmoid Colon of Autopsied Parkinson's Disease and Control Subjects..
LINK
https://doi.org/


a. Adjacent sections from a healthy control stained for phosphorylated α-Syn (MJFR-13, abcam ab168381) are shown with (left) and without (right) proteinase K treatment demonstrating robust detection of insoluble LP. b. Higher power view of Lewy Pathology (solid arrow) in a PD myenteric plexus ganglion. Samples obtained from Banner Health Brain and Body Donation Program. Scale bars are 50μm.

Acknowledgments
We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona, for the provision of human colon tissue and data. The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinsons Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimers Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimers Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinsons Disease Consortium) and the Michael J. Fox Foundation for Parkinsons Research.

We wish to acknowledge Yunfeng Li and the Translational Pathology Core Laboratory (TPCL) at UCLA for assistance with tissue processing, IHC staining, and scanning.
Materials


Note
ReagentBOND Polymer Refine Detection KitLeica BiosystemsCatalog #DS9800 contains
  • Peroxide Block (30 mL) 3–4% (v/v) Hydrogen peroxide.
  • Polymer (30 mL) Anti-rabbit Poly-HRP-IgG (<25 μg/mL) containing 10% (v/v) animal serum in tris-buffered saline/0.1% ProClin‱ 950.
  • DAB Part 1 (2.4 mL) 66 mM 3,3’-Diaminobenzidine tetrahydrochloride hydrate, in a stabilizer solution.
  • DAB Part B (30 mL) ≤0.1% (v/v) Hydrogen Peroxide in a stabilizer solution.
  • Hematoxylin (30 mL) <0.1% Hematoxylin



Equipment
new equipment
NAME
Leica BOND RX
BRAND
3342171
SKU
https://www.leicabiosystems.com/ihc-ish-fish/ihc-ish-instruments/products/leica-bond-rx/
SPECIFICATIONS

Protocol materials
ReagentBondTM Wash Solution 10X ConcentrateLeica BiosystemsCatalog #AR9590
In 2 steps
ReagentEnVision System-HRP, Labelled Polymer (Rabbit) Agilent TechnologiesCatalog #K4003
Step 7
ReagentBOND Polymer Refine Detection KitLeica BiosystemsCatalog #DS9800
Materials
ReagentBOND Enzyme Pretreatment KitLeica BiosystemsCatalog #AR9551
Step 2
ReagentEpitope Retrieval Solution 1 BONDLeica BiosystemsCatalog #AR9961
Step 2
ReagentAnti-Alpha-synuclein (phospho S129) antibody [MJF-R13 (8-8)]AbcamCatalog #AB168381
Step 5
ReagentHisto-ClearNational DiagnosticsCatalog #HS2001GLL
Step 17
ReagentChemical Permount Mounting MediumFisher ScientificCatalog #SP15-100
Step 17
ReagentDewax SolutionLeica BiosystemsCatalog #AR9222
Step 1
Leica Bond RX routine factory based “Bake and Dewax” protocol.ReagentDewax SolutionLeica BiosystemsCatalog #AR9222
Enzyme Antigen Retrieval: Incubate in Enzyme 1 solution for Duration00:20:00 at TemperatureRoom temperature
ReagentBOND Enzyme Pretreatment KitLeica BiosystemsCatalog #AR9551

Note
For comparison, this step can be replaced with Heat Induced Antigen Retrieval using ER1 buffer for Duration00:20:00
ReagentEpitope Retrieval Solution 1 BONDLeica BiosystemsCatalog #AR9961

20m
Incubate in Peroxide Block for Duration00:05:00
5m
Incubate in Bond Wash buffer three times Duration00:00:00 each.
ReagentBondTM Wash Solution 10X ConcentrateLeica BiosystemsCatalog #AR9590
Primary antibody: Incubate in 1:10,000
ReagentAnti-Alpha-synuclein (phospho S129) antibody [MJF-R13 (8-8)]AbcamCatalog #AB168381 for Duration01:00:00 at TemperatureRoom temperature
1h
Incubate in Bond Wash buffer three times Duration00:00:00 each.
ReagentBondTM Wash Solution 10X ConcentrateLeica BiosystemsCatalog #AR9590
Incubate with labeled polymer Duration00:10:00
ReagentEnVision+ System-HRP, Labelled Polymer (Rabbit) Agilent TechnologiesCatalog #K4003
10m
Incubate in Bond Wash buffer three times Duration00:02:00 each.
2m
Incubate with polymer for Duration00:08:00 .
8m
Incubate in Bond Wash buffer five times Duration00:00:00 each.
Deionized water wash Duration00:00:00 .
Incubate with Mixed DAB Refine for Duration00:00:00 followed by Mixed DAB Refine for Duration00:10:00 .
10m
Deionized water wash for three times Duration00:00:00 each.
Incubate with Hematoxylin for Duration00:10:00 .
10m
Incubate in Bond Wash buffer three times Duration00:00:00 each.
Deionized water wash Duration00:00:00 .
Slides are dehydrated in series of alcohols, cleared with ReagentHisto-ClearNational DiagnosticsCatalog #HS2001GLL and mounted with ReagentChemical Permount Mounting MediumFisher ScientificCatalog #SP15-100

Protocol references
Beach TG, Corbillé AG, Letournel F, Kordower JH, Kremer T, Munoz DG, Intorcia A, Hentz J, Adler CH, Sue LI, Walker J, Serrano G, Derkinderen P. Multicenter Assessment of Immunohistochemical Methods for Pathological Alpha-Synuclein in Sigmoid Colon of Autopsied Parkinson's Disease and Control Subjects. J Parkinsons Dis. 2016 Oct 19;6(4):761-770. doi: 10.3233/JPD-160888. PMID: 27589538; PMCID: PMC5501392.
Citations
Beach TG, Corbillé AG, Letournel F, Kordower JH, Kremer T, Munoz DG, Intorcia A, Hentz J, Adler CH, Sue LI, Walker J, Serrano G, Derkinderen P. Multicenter Assessment of Immunohistochemical Methods for Pathological Alpha-Synuclein in Sigmoid Colon of Autopsied Parkinson's Disease and Control Subjects.
https://doi.org/