Dec 09, 2024
Protocol for Multiplexed Immunofluorescence Imaging and Analysis of the Tumor Microenvironment
- 1Avantyx
Protocol Citation: Thomas S Lisse 2024. Protocol for Multiplexed Immunofluorescence Imaging and Analysis of the Tumor Microenvironment. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr93b3vmk/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: December 08, 2024
Last Modified: December 09, 2024
Protocol Integer ID: 114582
Abstract
High throughput multiplex immunofluorescent (mIF) image preparation and analysis provides a
rapid, dependable, and informative method for clinical diagnosis and prognosis
of diseases and therapeutic responses. This protocol describes the preparation
of a melanoma mIF panel designed to detect six protein biomarkers along with
DAPI in formalin-fixed paraffin-embedded (FFPE) human melanoma tumor sections. The
method involves sequential staining of tissue sections using the Leica BOND‱ RX
autostainer through repeated cycles of heat-induced epitope retrieval (HIER),
blocking, primary antibody incubation, secondary antibody binding, Opal
fluorophore application, and antibody elution. The Opal fluorescent signal
remains stable during the antibody stripping process, enabling additional
rounds of staining for new targets without the risk of cross-reactivity, even
for co-localized markers. The protocol also incorporates nuclear
counterstaining with DAPI. While the protocol accommodates up to seven markers
(six antigens plus DAPI), individual antigen targets can be substituted and
re-optimized as needed. The outlined workflow includes the following steps: (1)
Staining using the Leica Bond‱ RX, (2) Scanning with the Akoya PhenoImager HT‱
2.0, and (3) image annotation and analysis using PhenoChart 3.0, inFORM‱ 3.0, and
phenoptrReports 0.3.2.
This protocol leverages multispectral imaging and built-in spectral unmixing to
facilitate efficient large-scale spatial signature analysis. The chosen
antibodies—FoxP3, PD-L1, Sox10, PD-1, CD8, and CD68—enable a thorough
assessment of PD-L1- and PD-1-mediated immune responses in the context of
melanoma tumor cells.
Attachments
Guidelines
Guidelines from the CDC and CAP outline safe work practices for human medical and histopathological
diagnostic laboratories. Any specimen suspected of containing tubercle bacilli
must be processed in a BSL-3 laboratory using Class IIA2 biological safety cabinets
that undergo annual inspections. Once the sample has been inactivated or its
genetic material extracted, subsequent testing—including multiplex
immunofluorescence (mIF) processing and data analysis—can be safely conducted
in a BSL-2 laboratory or office environment.
Materials
Materials and Reagents
Antibodies
Item Company Catalog Number
Anti-FoxP3
D608R Cell Signaling Technology #12653
Anti-PD-L1
(SP263) Roche Diagnostics 790-4905
Anti-Sox10
EP268-1 Cell Marque 383R-14
Anti-PD-1
EPR4877(2) Abcam ab137132
Anti-CD8
4B11 Leica
Biosystems CD8-4B11-L-U
Anti-CD68
PG-M1 Thermo Fisher Scientific MA5-12407
mIF
Processing and Labeling Reagents
Item Company Product #
1X Plus Auto Amplification Diluent Akoya
Biosciences FP1609
Opal Polymer HRP Ms + Rb Akoya Biosciences ARH1001EA
Antibody Diluent/Block buffer Akoya Biosciences ARD1001EA
Opal
480 Reagent Akoya
Biosciences FP1500001KT
Opal
520 Reagent Akoya
Biosciences FP1487001KT
Opal
540 Reagent Akoya
Biosciences FP1494001KT
Opal
570 Reagent Akoya
Biosciences FP1488001KT
Opal
620 Reagent Akoya
Biosciences FP1495001KT
Opal
650 Reagent Akoya
Biosciences FP1496001KT
Opal
690 Reagent Akoya Biosciences FP1497001KT
Opal
780 Reagent Pack Akoya Biosciences FP1501001KT
(Opal
TSA-DIG, 1 vial,
Opal
Polaris 780, 1 vial)
Spectra
DAPI Akoya
Biosciences FP1501001KT
Instruments
Item Company Catalog Number
BOND
RX Automated Stainer Lecia Biosystems 21.2821
PhenoImager
HT‱ 2.0 Automated Quantitative Akoya
Biosystems CLS143455
Pathology
Imaging System
Bond
RX Reagents and Materials
Item Company Catalog Number
Titration
Kit Leica Biosystems OPT9049
Open
container - 30 mL Leica Biosystems OP309700
Titration
kit container - 6 mL Leica Biosystems OPT9049
Research
Detection System 2 Leica Biosystems DS9777
Universal
Covertiles Leica Biosystems S21.4611
Slide
Tray Leica Biosystems S21.0304
Reagent
Tray Leica Biosystems S21.1003
Slide
Labels and Printer Ribbon Leica Biosystems S21.4564
Apex
Adhesive Slide Leica Biosystems 3800040
Dewax
Solution Leica
Biosystems AR9222
Epitope
Retrieval Solution 1 (pH6) Leica
Biosystems AR9961
Epitope
Retrieval Solution 2 (pH9) Leica
Biosystems AR9640
BOND
Wash Solution 10X Concentrate Leica Biosystems AR9590
BOND
Aspirating Probe Cleaning System Leica
Biosystems CS9100
Coverslip
24 x 32 mm #1 (0.13-0.16 mm) Fisher
Scientific 15717592
ProLong‱
Diamond Antifade Mountant Thermo Fisher Scientific P36965
Data
Analysis
Item Company
inFORM‱
software 3.0 Akoya
Biosciences
PhenoChart‱
2.0 Akoya Biosciences
phenoptrReports
0.3.2 Akoya Biosciences
PhenoImager
HT‱ 2.0 Akoya Biosciences
R
4.4.2 open source
RStudio
2024.09.1+394 open
source
FFPE
Melanoma Tissue Slides
Item Company Catalogue
Number
Human
Skin Tumor: Malignant Melanoma BioChain T2235218-3
Human
Adult Normal: Tonsil BioChain T2234268