Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Podsumowanie

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

Streszczenie

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

Wprowadzenie

Cats with hypertrophic cardiomyopathy are at risk of life-threatening thromboembolic complications^1,2. Despite the high morbidity and mortality associated with feline cardiogenic arterial thromboembolism (CATE), the underlying pathophysiology of CATE in cats is poorly understood. There are also limited diagnostic and therapeutic tools to treat and identify cats at risk of this devastating condition³.

In addition to its role in innate immunity, neutrophils have been shown to play a role in thrombosis by releasing neutrophil extracellular traps (NETs), which are web-like networks of cell-free DNA (cfDNA) encrusted with histones and granular proteins like neutrophil elastase (NE) and myeloperoxidase. Neutrophils undergo NETs formation in response to systemic inflammation, direct encounter with pathogens, and interaction with activated platelets^4,5,6,7. In dogs, neutrophil-derived DNA has been shown to inhibit clot lysis, while NET proteins accelerate clot formation. The ability of NETs to trap circulating cells and coagulation components is also key to their thrombogenic properties^8,9,10,11,12.

NETs are detected by colocalization of extracellular neutrophil proteins, histones, and cfDNA. Because of this, the identification and quantification of NETs in fixed tissues by immunofluorescence of deparaffinized tissues is superior to traditional hematoxylin and eosin (H&E) stain using bright field microscopy^4,5. Several human studies using immunofluorescence microscopy identified NETs as structural components of coronary arterial thrombi, cerebral stroke thrombi, atherothrombosis, and venous thrombi^{13,14,15,16,17}. To date, a standardized method to detect and quantify NETs in feline thrombi has not been described. Because the identification of NETs in feline cardiogenic arterial thrombi may facilitate future translational research in NETs and thrombosis, we describe techniques of NET identification and assessment in paraffin-embedded arterial thrombi in cats.

Protokół

All methods described here were performed in accordance to the guidelines of the Institutional Animal Care and Use Committee at the University of California, Davis. Necropsies and biopsies of tissues were performed with owners’ consent.

1. Tissue fixation, embedding, and sectioning

1. Dissect out the aortic bifurcation, including the descending aorta, femoral artery, and the common iliac arteries (Figure 1A), shortly after humane euthanasia or death. Blunt dissect out the fascia (Figure 1B) before submerging it completely in 10% neutral-buffered formalin for a minimum of 24 h and no longer than 48 h.
2. To dehydrate the sample, first submerge in 10% neutral-buffered formalin heated to 37 °C for 1 h. Then, submerge in increasing concentrations of ethanol heated to 37 °C (70%, 95%, 100%) 2x for 1 h each. Finally, without rinsing, submerge 2x in 100% toluene heated to 37 °C for 1 h each.
3. Add paraffin heated to 62 °C and allow the paraffin to solidify completely overnight.
4. Section 2–3 µm of the paraffin-embedded tissue using a microtome and place on positively charged glass slides. Store sectioned tissues at -80 °C until further analysis.

2. Deparaffinization, rehydration, and heat-induced antigen retrieval

1. To perform deparaffinization and rehydration of sections on glass slides, place glass slides in racks and process in the following order:
   1. Submerge completely in 100% xylene for 3 min. Repeat this step 2x. Do not rinse in between steps.
   2. Submerge completely in decreasing concentrations of ethanol (100%, 95%, 70%) at room temperature (RT), 3x for 3 min each. Do not rinse in between steps.
   3. Submerge completely in deionized water for 2 min. Repeat.
2. Place sections into Tris-buffered saline with 0.1 % Tween (TBST, pH = 7.6) for 2–3 min.
3. Fill the reservoir with deionized water heated to 100 °C. Allow the steamer chamber to equilibrate for 20 min.
   NOTE: Heat-induced antigen retrieval is best performed with indirect heating generated by a steamer with a preset temperature setting, such as a food steamer.
4. Heat the commercially available antigen retrieval solution containing Tris and EDTA (pH = 9) to 95–97 °C on a temperature-controlled hot plate with constant stirring. Ensure that it does not boil.
   NOTE: The solution should turn cloudy once it is warmed.
5. Pour the heated antigen retrieval solution into a slide container and place the container in the chamber of the steamer. Allow the antigen retrieval solution to equilibrate to the temperature of the steamer for 3–4 min. Ensure that the temperature of the chamber is ~95 °C.
6. Submerge the slides completely in the heated antigen retrieval solution and continue the application of external heating via the steamer for 20 min.
7. Remove the slide container from the steamer and allow the slides and the antigen retrieval solution to cool to RT. Store the diluted antigen retrieval solution at 4 °C and reuse up to 2x if needed.
8. Wash the slides 3x with TBST for 5 min.

3. Immunolabeling and autofluorescence quenching

NOTE: Table 1 details the composition of the blocking buffers used in the following steps.

1. Incubate sections in Blocking Buffer 1 for 2 h at RT under gentle rocking (30–50 rpm). Seal with paraffin film to avoid drying.
2. Without washing, immediately apply 100 µL of diluted rabbit polyclonal anti-human citrullinated histone H3 (citH3) antibody (0.03 mg/mL diluted in blocking buffer 1) directly onto the slide.
3. Place a coverslip (24 mm x 40 mm x 0.13–0.17 mm) on each section to allow even distribution of the antibody mixture.
4. Incubate for 12–16 h at 4 °C with gentle rocking (30–50 rpm). Seal with parafilm film to avoid drying.
5. Wash 3x with TBST for 5 min.
6. Apply 100 µL of goat anti-rabbit antibody conjugated to Alexa Fluor 488 (diluted to a final concentration of 0.04 mg/mL or 1:50 in Blocking Buffer 1) as described in step 3.3. Incubate for 1 h at RT under gentle rocking (30–50 rpm). Protect slides from light.
7. Wash with TBST 3x for 5 min.
8. Incubate sections in Blocking Buffer 2 overnight at 4 °C under gentle rocking (30–50 rpm). Protect from light.
9. Wash with TBST 3x for 5 min.
10. Block sections in Blocking Buffer 3 as described in step 3.3 at RT for 2 h under gentle rocking (30–50 rpm).
11. Incubate sections with biotinylated polyclonal rabbit anti-human NE antibody (final concentration = 0.2 µg/mL in Blocking Buffer 3) at 4 °C for 12–16 h as described in steps 3.2–3.4.
12. Wash with TBST 3x for 5 min.
13. Incubate with Alexa Fluor 594 streptavidin conjugate (dilute to 1:100 or 0.02 mg/mL in Blocking Buffer 3) as described in steps 3.2–3.3 for 1 h at RT. Protect from light and seal with paraffin to prevent drying.
14. Wash with TBST 1x for 5 min.
15. Apply 100 µL of autofluorescence quenching solution mixture directly onto the sections for 1 min as instructed by the manufacturer.
16. Immediately wash the slides with TBST 6x for 10 min.
17. Cover each slide with 100 µL of 300 nM DAPI for 5 min in the dark.
18. Wash with TBST for 3 min. Repeat this for a total of 5x.
19. Apply a drop (~50 μL) of antifade mounting medium, part of the autofluorescence quenching kit, directly onto the glass slide surrounding the section. Place a coverslip (24 mm x 40 mm x 0.13–0.17 mm) gently onto the section without creating any bubbles.
20. Allow samples to cure overnight in the dark at 4 °C until the mounting medium has hardened for microscopic analysis with immersion lenses.

4. Neutrophil extracellular trap identification

NOTE: The following protocol utilizes an inverted epifluorescence microscope with a 1,280 x 960 digital CCD camera (see Table of Materials).

1. To locate thrombi, scan cranially to caudally along the length of the aorta, aortic bifurcation, and each femoral artery using phase contrast microscopy with a 10x objective. A thrombus is a conglomeration of tissue containing red blood cells, white blood cells, and platelets adjacent to the endothelium on phase contrast and bright field microscopy (Figure 2A, Figure 2B).
2. First examine sections for NETs using the DAPI channel (excitation = 357/44 nm) with 10x and 20x objectives (Figure 2C). Note that cfDNA appears as decondensed DNA that is not within the confines of the cytoplasm of a cell when seen on phase contrast or bright field microscopy.
3. Identify extracellular NE and citH3 on the Texas Red channels (excitation = 585/29 nm, emission = 628/32 nm) and green fluorescent protein channel (excitation = 470/22 nm, emission = 525/50 nm), respectively with 10, 20, and 40x objectives.
4. Evaluate and analyze NETs within a thrombus using available software, such as Image J (NIH). NET formation is identified based on the colocalization of cfDNA, extracellular citH3, and NE as previously described¹⁸. Maintain consistent exposure time and gains of each channel throughout the acquisition of images to avoid saturation in pixel intensity.
5. Map each thrombus based on its proximity to the descending aorta by dividing it into three equal zones, with Zone 1 closest to the aorta, Zone 3 furthest from the aorta, and Zone 2 between Zones 1 and 3). With the operator blinded to the medical condition of each subject, take at least ten random fields in each zone. Characterize the distribution of NETs in thrombi by averaging the numbers of fields with NETs in each zone or calculating the average NET-occupying area per zone.

Wyniki

Using this protocol for deparaffinization, heat-induced antigen retrieval, and double immunolabeling of paraffin-embedded thrombi, we identified NETs in feline CATE for the first time. Thrombi within the aortic bifurcation were located by fluorescence microscopy and bright field microscopy using standard H&E staining and phase contrast microscopy. On bright field microscopy, feline arterial thrombi consisted of red blood cells, leukocytes, fibrin, and platelets (Figure 3A). Although H...

Dyskusje

We describe a protocol to identify NETs in fixed feline cardiogenic arterial thrombi using a double immunolabeling protocol and immunofluorescence microscopy. Although only cardiogenic arterial thrombi were stained, in theory this protocol could be used for other types of thrombi and in other veterinary species. Identification of NETs within feline arterial thrombi suggests that NETs may play a role in thrombosis in cats.

Detection of NETs by immunofluorescence in fixed and paraffin-embedded t...

Materiały

Name	Company	Catalog Number	Comments
4,6-Diamidino-2-phenylin (DAPI)	Life Technologies Corporation	D1306
Alexa Fluor 594 Streptavidin conjugate	ThermoFisher Scientific	Catalog # S11227
Anti-citrullinated histone H3 antibody	Abcam	Ab5103
EVOS FL Cell Imaging System	ThermoFisher Scientific	AMEFC4300
EVOS Imaging System Objective 10x	ThermoFisher Scientific	AMEP4681	NA 0.25, WD 6.9/7.45 mm
EVOS Imaging System Objective 20x	ThermoFisher Scientific	AMEP4682	NA 0.40, WD 6.8 mm
EVOS Imaging System Objective 40x	ThermoFisher Scientific	AMEP4699	NA 0.75, WD 0.72 mm
Goat anti-rabbit Alexa Fluor 488 antibody	ThermoFisher Scientific	Catalog # A32723
Goat serum	Jackson Immuno Research Labs	Catalog # NC9660079. Manufacturer Part # 005-000-121
Neutrophil elastase antibody	Bioss Antibodies	Bs-6982R-Biotin	Rabbit polyclonal Antibody, Biotin conjugated
NP40	Pierce	Product # 28324. Lot # EJ64292
Positive charged microscope slides	Thomas Scientific	Manufacturer No. 1354W-72
Rabbit serum	Life Technology	Catalog # 10510
Target Retrieval Solution	Agilent Dako	S2367	TRIS/EDTA, pH 9 (10x)
TrueVIEW Autofluorescence Quenching Kit	Vector Laboratories	SP-8400