Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

Summary

Despite the advancements in multiplex immunohistochemistry and multispectral imaging, characterizing the density and clustering of major immune cells simultaneously in the endometrium remains a challenge. This paper describes a detailed multiplex staining protocol and imaging for the simultaneous localization of four immune cell types in the endometrium.

Abstract

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical diagnostics. It can be used to characterize various biological processes or pathologies, such as wound-healing, immune response, tissue rejection, and tissue-biomaterial interactions. However, the visualization and quantification of multiple antigens (especially for immune cells) in a single tissue section using conventional immunohistochemical (IHC) staining remains unsatisfactory. Hence, multiplexed technologies were introduced in recent years to identify multiple biological markers in a single tissue sample or an ensemble of different tissue samples.

These technologies can be especially useful in differentiating the changes in immune cell-to-cell interactions within the endometrium between fertile women and women with recurrent miscarriages during implantation. This paper describes a detailed protocol for multiplexed fluorescence IHC staining to investigate the density and clustering of four major immune cell types simultaneously in precisely timed endometrial specimens during embryo implantation. The method includes sample preparation, multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis, with specific reference to detecting endometrial immune cells.

Using this method, the density and clustering of four major immune cell types in the endometrium can be simultaneously analyzed in a single tissue section. In addition, this paper will discuss the critical factors and troubleshooting to overcome possible fluorophore interference between the fluorescent probes being applied. Importantly, the results from this multiplex staining technique can help provide an in-depth understanding of the immunologic interaction and regulation during embryo implantation.

Introduction

Recurrent miscarriage (RM) can be defined as the loss of two or more pregnancies before 24 weeks of gestation¹. This frequent reproductive condition affects up to 1% of couples worldwide^2,3. The pathophysiology is multifactorial and can be divided into embryologically driven causes (mainly due to an abnormal embryonic karyotype) and maternally driven causes that affect the endometrium and/or placental development. This manifestation can result from parental genetic abnormalities, uterine anomalies, prothrombotic conditions, endocrinology factors, and immunological disorders

Protocol

The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Informed consent was obtained from the participants before collecting the endometrial biopsies. Refer to the introduction section for inclusion criteria of the control and RM groups.

1. Sample preparation

1. Ensure that all the women in this study undergo a daily urine dipstick test from day 9 of the menstrual cycle onwards to identify the luteinizing hormone (LH) surge to detect ovulation, and time the endometrial biopsies precisely on the 7^th day after the LH surge (LH+7).
2. Obtai....

Results

The overall schematic process of performing a 4-color multiplex assay for the detection of 4 endometrial immune cell types is shown in Figure 1. In brief, the protocol for this multiplex immunofluorescence staining required 8 key steps: 1. Slide preparation, 2. Epitope retrieval, 3. Blocking, 4. Primary antibody application, 5. Secondary antibody application, 6. Signal amplification, 7. Removal of antibody, and 8. Counterstain and mount. Image rendering and analysis were then conducted using.......

Discussion

Critical steps within the protocol
It is important to note that multiplex staining requires diligent optimization. Antigen retrieval, using citrate buffer and microwave technology, requires optimization to ensure complete antibody stripping and maintain tissue viability. As TSA reagents covalently bind to sites surrounding the antigen, they can potentially inhibit the binding of a subsequent primary antibody through steric hindrance (also known as the "umbrella effect"). This tends to occur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amplification Diluent	Perkin Elmer	FP1498	Fluorophore diluent buffer
Antibody diluent	Perkin Elmer	ARD1001EA	Diluting the antibody
CD3	Spring Bioscience	M3072	Primary antibody
CD20	Biocare Medical	CM004B	Primary antibody
CD56	Leica	NCL-CD56-504	Primary antibody
CD68	Spring Bioscience	M5510	Primary antibody
Citrate Buffer Solution, pH 6.0 (10x)	Abcam	AB64214	Antigen retrieval solution
EMSURE Xylene (isomeric mixture)	Merck	108297	Dewaxing
Ethanol absolute	Merck	107017	Ethyl alcohol for rehydration
HistoCore BIOCUT Manual Rotary Leica Microtome	Leica	RM2125RTS	Sectioning of paraffin-embedded tissue
inForm Advanced Image Analysis Software	Perkin Elmer	inForm® Tissue Finder Software 2.2.1 (version 14.0)	Data Analysis software
Mantra® Workstation	Akoya Biosciences	CLS140089	Spectral imaging
Microwave	Panasonic	Inverter	Microwave stripping
Opal 520	Perkin Elmer	FP1487A	Appropriate tyramide based fluorescent reagent
Opal 620	Perkin Elmer	FP1495A	Appropriate tyramide based fluorescent reagent
Opal 650	Perkin Elmer	FP1496A	Appropriate tyramide based fluorescent reagent
Opal 690	Perkin Elmer	FP1497A	Appropriate tyramide based fluorescent reagent
Oven	Memmert	U10	Dewaxing
Peroxidase Blocking Solution	DAKO	S2023	Removal of tissue peroxidase activities
Poly-L-lysine coated slide	FISHER SCIENTIFIC	120-550-15	Slide for routine histological use
PolyHRP Broad Spectrum	Perkin Elmer	ARH1001EA	Secondary antibody
ProLong™ Gold Antifade Mountant	ThemoFisher Scientific	P36930	Mounting
Spatstat	/	Version 2.1-0	Spatial point pattern analysis
Spectral DAPI	Perkin Elmer	FP1490A	Nucleic acid staining
Tissue Processor	Thermo Fischer	Excelsior ES	Tissue processing for dehydration and paraffination
Tris Buffer Saline (TBS), 10x	Cell Signaling Technology	12498S	Washing solution
Tween 20	Sigma-Aldrich	P1370-1L	Nonionic detergent