Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system

Research output: Contribution to journalArticle

Abstract

The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

Original languageEnglish
Pages (from-to)25-34
Number of pages10
JournalJournal of Immunological Methods
Volume472
DOIs
Publication statusPublished - 1 Sep 2019

Fingerprint

Fluorescent Antibody Technique
Fluorescence
Color
Immunophenotyping
Paraffin
Formaldehyde
Immunohistochemistry
T-Lymphocytes
Research

Cite this

@article{89079d71e8e74abaaa63c85a7a5aa5c9,
title = "Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system",
abstract = "The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.",
keywords = "Colorectal, Fluorescence microscopy, Imaging, Immunohistochemistry, T cells",
author = "Anyaegbu, {Chidozie C.} and Lee-Pullen, {Tracey F.} and Miller, {Timothy J.} and Abel, {Tamara N.} and Platell, {Cameron F.} and McCoy, {Melanie J.}",
year = "2019",
month = "9",
day = "1",
doi = "10.1016/j.jim.2019.06.011",
language = "English",
volume = "472",
pages = "25--34",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",

}

TY - JOUR

T1 - Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system

AU - Anyaegbu, Chidozie C.

AU - Lee-Pullen, Tracey F.

AU - Miller, Timothy J.

AU - Abel, Tamara N.

AU - Platell, Cameron F.

AU - McCoy, Melanie J.

PY - 2019/9/1

Y1 - 2019/9/1

N2 - The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

AB - The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

KW - Colorectal

KW - Fluorescence microscopy

KW - Imaging

KW - Immunohistochemistry

KW - T cells

UR - http://www.scopus.com/inward/record.url?scp=85067097097&partnerID=8YFLogxK

U2 - 10.1016/j.jim.2019.06.011

DO - 10.1016/j.jim.2019.06.011

M3 - Article

VL - 472

SP - 25

EP - 34

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

ER -