Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Summary

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Abstract

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 µm thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.

Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Introduction

Neutrophil extracellular traps (NETs) were first visualized by Brinkmann et al. as a pathway of cellular death different from apoptosis and necrosis in 2004¹. In this pathway, neutrophils release their decondensed chromatin into the extracellular space to form large web-like structures covered in antimicrobial proteins that were formerly stored in the granules or cytosol. These antimicrobial proteins include neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (H3cit), which are commonly used for indirect immunofluorescence detection of NETs². This method not only identifies the quantitative pres....

Protocol

This study included mouse tissues derived from experiments approved by the Hamburg State Administration for Animal Research, Behörde für Justiz und Verbraucherschutz, Hamburg, Germany (73/17, 100/17, 94/16, 109/2018, 63/16). The tissues used were mouse lung and colon from a septic model and burned skin. We used 8 week old male and female mice. The European Directive 2010/63/EU on the protection of animals used for scientific purposes was followed for all the experiments. The anonymized human samples included tissues from neonatal enterocolitis, burned skin, biliary atresia, spondylodiscitis, and myocardium. According to the Medical Research Ethics Committee ....

Results

Before starting our protocol optimization, we identified key steps for successful staining by searching PubMed for studies that used FFPE tissue for the immunostaining of NETs and compared their protocols. The most promising protocol differences were identified as the key steps for the protocol optimization, while steps that mostly corresponded to each other were not changed (Table 1).

Table 1: PubMed Research for FFPE immunostaining of NETs. This table shows .......

Discussion

In this work, we aimed to adapt and optimize the existing protocols for imaging NETs to more tissue types, beginning with the actual staining process. The first critical step for this method is the selection of the most suitable antibodies. For NE, we tried an NE antibody from a mouse host on human tissue, which showed no reliable staining compared to NE from a rabbit host. Furthermore, Thålin et al. proposed H3cit (R8) as a more specific antibody for extracellular staining. We compared this antibody with the widely used.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
			Dilution
Anti-Neutrophil Elastase antibody 100µg	abcam	Ab 68672	1:100
Anti-Histone H3 (citrulline R2 + R8 + R17) antibody  100µg	abcam	Ab 5103	1:50
Anti-Myeloperoxidase antibody [2C7] anti-human 100 µg	abcam	Ab 25989	1:50
Anti-Myeloperoxidase antibody [2D4] anti-mouse 50 µg	abcam	Ab 90810	1:50
Axiovision Microscopy Software	Zeiss	4.8.2.
Blocking solution with donkey serum (READY TO USE) 50ml	GeneTex	GTX30972
Coverslips	Marienfeld	0101202
Dako Target Retrieval Solution Citrate pH6 (x10)	Dako	S2369
DAPI 25 mg	Roth	6335.1	1:25000
DCS antibody dilution 500 mL	DCS diagnostics	DCS AL120R500
Donkey ant goat Cy3	JacksonImmunoResearch	705-165-147	1:200
Donkey anti rabbit AF647	JacksonImmunoResearch	711-605-152	1:200
Donkey anti rabbit Cy3	JacksonImmunoResearch	711-165-152	1:200
Fluoromount-G Mounting Medium	Invitrogen	00-4958-02
Glass slide rack	Roth	H552.1
Human/Mouse MPO Antibody	R&D Systems	AF 3667	1:20
Hydrophobic Pen	KISKER	MKP-1
Isokontrolle Rabbit IgG Polyclonal 5mg	abcam	Ab 37415	1:2000 and 1:250
MaxBlock Autofluorescence Reducing Reagent Kit (RUO) 100 ml	Maxvision	MB-L
Microscopy camera	Zeiss	AxioCamHR3
Microwave	Bosch	HMT84M421
Mouse IgG1 negative control	Dako	X0931 Aglient	1:50 and 1:5
Normal Goat IgG Control	R&D Systems	AB-108-C	1:100
PBS Phosphate buffered saline (10x)	Sigma-Aldrich	P-3813
PMP staining jar	Roth	2292.2
Recombinant Anti-Histone H3 (citrulline R8) antibody 100µg	abcam	Ab 219406	1:100
Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control 200µg	abcam	Ab 172730	1:300
ROTI Histol	Roth	6640
SuperFrost Plus slides	R. Langenbrinck	03-0060
TBS Tris buffered saline (x10)	Sigma-Aldrich	T1503
Triton X-100	Sigma-Aldrich	T8787
Tween 20	Sigma-Aldrich	P9416
Water bath	Memmert	830476
Water bath rice cooker	reishunger	RCP-30
Wet chamber	Weckert Labortechnik	600016
Zeiss Widefield microscope	Zeiss	Axiovert 200M