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.
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.
Cats with hypertrophic cardiomyopathy are at risk of life-threatening thromboembolic complications1,2. 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 platelets4,5,6,7. 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 properties8,9,10,11,12.
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 microscopy4,5. Several human studies using immunofluorescence microscopy identified NETs as structural components of coronary arterial thrombi, cerebral stroke thrombi, atherothrombosis, and venous thrombi13,14,15,16,17. 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.
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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
- 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.
- 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.
- Add paraffin heated to 62 °C and allow the paraffin to solidify completely overnight.
- 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
- To perform deparaffinization and rehydration of sections on glass slides, place glass slides in racks and process in the following order:
- Submerge completely in 100% xylene for 3 min. Repeat this step 2x. Do not rinse in between steps.
- 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.
- Submerge completely in deionized water for 2 min. Repeat.
- Place sections into Tris-buffered saline with 0.1 % Tween (TBST, pH = 7.6) for 2–3 min.
- 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.
- 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.
- 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.
- Submerge the slides completely in the heated antigen retrieval solution and continue the application of external heating via the steamer for 20 min.
- 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.
- 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.
- Incubate sections in Blocking Buffer 1 for 2 h at RT under gentle rocking (30–50 rpm). Seal with paraffin film to avoid drying.
- 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.
- 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.
- Incubate for 12–16 h at 4 °C with gentle rocking (30–50 rpm). Seal with parafilm film to avoid drying.
- Wash 3x with TBST for 5 min.
- 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.
- Wash with TBST 3x for 5 min.
- Incubate sections in Blocking Buffer 2 overnight at 4 °C under gentle rocking (30–50 rpm). Protect from light.
- Wash with TBST 3x for 5 min.
- Block sections in Blocking Buffer 3 as described in step 3.3 at RT for 2 h under gentle rocking (30–50 rpm).
- 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.
- Wash with TBST 3x for 5 min.
- 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.
- Wash with TBST 1x for 5 min.
- Apply 100 µL of autofluorescence quenching solution mixture directly onto the sections for 1 min as instructed by the manufacturer.
- Immediately wash the slides with TBST 6x for 10 min.
- Cover each slide with 100 µL of 300 nM DAPI for 5 min in the dark.
- Wash with TBST for 3 min. Repeat this for a total of 5x.
- 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.
- 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).
- 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).
- 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.
- 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.
- 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.
- 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.
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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&E cannot stain specific NET components, NETs frequently appeared as networks of deep purple threads of various lengths surrounding nearby erythrocytes and leukocytes (Figure 3A, dotted outline). A thrombus was characterized as a well demarcated structure within the vascular space on phase contrast microscopy (Figure 2A, Figure 4B). We further confirmed the presence of NETs within these areas by immunofluorescence microscopy (Figure 3B). Magnification of these areas revealed large aggregates of NETs, composed of cfDNA, extracellular citH3, and NE (Figure 2C, Figure 3B, white dotted outline). Using the same technique to search for thrombi and NETs in cats without CATE, we found that sheets of lyzed neutrophils could be detected occasionally in close proximity to the endothelium. Although these neutrophils displayed some morphological characteristics of NET formation, they should not be associated with any organized thrombus. We did not identify any thrombi in any of the control samples (Figure 4A).
Figure 5A demonstrates profound autofluorescence of clot elements like erythrocytes and collagen when imaged at the green (488 nm) wavelength, which hindered our ability to detect cfDNA and protein colocalization. We found that brief autofluorescence quenching using a commercially available kit after immunolabeling significantly increased the sensitivity of protein colocalization and NET detection, even in areas with an abundance of erythrocytes (Figure 5B, arrowheads).
Figure 1: Representative necropsy photographs of a dissected aortic bifurcation from a cat with cardiogenic arterial thromboembolism. (A) The descending aorta was dissected 4–5 cm cranial (Cr) to the aortic bifurcation. (B) Fascia was carefully dissected out until the descending aorta (1) and iliac arteries (2,3) were clearly visible at the caudal aspect (Cd). Note the thrombus within the aortic bifurcation (*). Please click here to view a larger version of this figure.
Figure 2: Representative phase contrast and immunofluorescence images of NETs in a thrombus found within a feline aortic bifurcation. (A) Phase contrast microscopy revealed a thrombus as a discrete and well demarcated structure close to the aorta. Combined phase contrast and fluorescence staining of DNA (blue) showed the presence of leukocytes and cell-free DNA within the thrombus. The boxed area consists of a large concentration of cell-free DNA. Original 10x magnification; Scale bar = 400 µm. (B) The boxed area in (A) was further magnified at 20x. Cell-free DNA and intracellular DNA stained with DAPI (blue), neutrophil elastase (NE), and citrullinated histone H3 (citH3) appeared green and red, respectively. (Original 20x magnification; Scale bar = 100 µm). (C) NETs, identified based on colocalization of cell-free DNA, extracellular NE, and citH3, were outlined (dotted line). Original 40x magnification; Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 3: Representative image of a feline arterial thrombus using H&E and immunofluorescence staining. (A) On H&E stain, large concentration of neutrophils and erythrocytes were visible. Extracellular chromatins appeared as deep purple threads of various lengths surrounded by erythrocytes and neutrophils (dotted outline, black arrow). (B) Neutrophil extracellular traps were easily visualized using immunofluorescence microscopy on the same thrombus (dotted outline, white arrow). NETs were identified as colocalization of cfDNA (blue), NE (green), and citH3 (red). Original 20x magnification; Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 4: Representative phase contrast and immunofluorescence images of aortic bifurcations. (A) Phase contrast and immunofluorescence images of aortic bifurcations in a cat without arterial thrombosis. Note the absence of thrombi or aggregates of neutrophils within the lumen of the aortic bifurcation from the cat without arterial thrombosis. (B) Phase contrast and immunofluorescence images of aortic bifurcations in a cat diagnosed with cardiogenic arterial thromboembolism. The aortic bifurcations were stained for DNA (blue), neutrophil elastase (green), and citrullinated histone H3 (red). In this case, a well demarcated thrombus bulging into the vascular wall and occupying most of the aortic lumen was noted in the cat with cardiogenic arterial thromboembolism. NETs, characterized by colocalization of cfDNA, NE, and citH3, were identified within the thrombus (dotted outline). Original 10x magnification; Scale bar = 400 µm. Please click here to view a larger version of this figure.
Figure 5: Representative phase contrast (PC) and immunofluorescence images of arterial thrombi from two cats. The slides were stained for citH3, NE, and DNA and imaged at 488 nm (red), 595 nm (green), and 357 nm (blue) wavelengths, respectively, at 40x magnification. Cardiogenic arterial thrombi in cats had an abundance of erythrocytes (*, dotted line). (A) Autofluorescence from erythrocytes was most prominent across the 488 nm wavelength, diminishing the detection of colocalization signal and identification of NETs. (B) Quenching significantly reduced autofluorescence at the 488 nm wavelength, especially in areas with a high concentration of erythrocytes (*, dotted line). It enhanced the detection of colocalized proteins, citH3, and neutrophil elastase (arrowhead), in the presence of erythrocytes (Scale bar = 200 µm). Please click here to view a larger version of this figure.
Supplement Figure 1: Representative immunofluorescence images of arterial thrombi from a cat. The sections were stained for DNA (blue), citH3 (red), and either myeloperoxidase (A) or neutrophil elastase (B). (A) Despite utilizing a feline-specific myeloperoxidase antibody (MPO, 1:5), the staining intensity of MPO remained poor. (B) Using a polyclonal neutrophil elastase (NE) antibody, known to cross-react with multiple species, the immunoreactivity and staining intensity were significantly higher. Note the characteristic lobulated nuclei of neutrophils surrounded by NE (arrows). Original 40x magnification; Scale bar = 100 µm. Please click here to download this figure.
|1||TBS with 0.1% Tween-20, 0.1% NP40, 5% goat serum|
|2||TBS with 0.1% Tween-20, 10% rabbit serum, 0.1%NP-40|
|3||TBS with 0.1% Tween-20, 5% BSA, 0.1% NP-40|
Table 1: Composition of blocking buffers used for immunofluorescence.
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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 tissue is superior to conventional histological stains like H&E, which often shows threads of chromatin surrounded by neutrophils13. Immunohistochemistry and immunofluorescence of fixed arterial thrombi allow the simultaneous detection of cfDNA and other extracellular proteins like citH3, known to be specific to NETs formation18,19. Because cryopreparations are suboptimal for NET detection in tissues and thrombi, we preserved our samples in 10% neutral-buffered formalin, which contains 4% formaldehyde and 10% methanol. Cell-free nucleic acids are not directly fixed by formaldehyde. Instead, they are immobilized within fixed protein structures, which are altered by partially reversible methylene bridge crosslinks induced by formaldehyde20. To further limit artefacts and autofluorescence caused by formic acid and ketones generated by oxidation of formaldehyde, investigators can choose to use methanol-free paraformaldehyde diluted in buffer for fixation. Because the duration of fixation affects immunoreactivity, we recommend fixation for no longer than 24 h prior to dehydration and paraffin embedding. Paraffin-embedded tissues or clots can then be stored for deparaffinization and staining.
Chemical fixation by formaldehyde and paraformaldehyde alters the tertiary structure of proteins, masking the antigens of interest and preventing the binding of antibodies to specific epitopes21. Antigen retrieval, a process that breaks the methylene bridge crosslinks, is essential prior to performing immunodetection in formalin-fixed tissues. Based on the authors’ experience, heat-induced antigen retrieval using an alkaline retrieval solution (pH = 9 in Tris/EDTA buffer) with mild indirect heating enhances the detection of proteins and NETs while minimizing artefacts and autofluorescence. The temperature of the antigen retrieval solution should not reach the boiling point (>100 ˚C), because the denaturing of proteins can lead to nonspecific binding and background noise.
A limitation of NET identification in fixed clots is that immunofluorescence staining can be highly variable under different antigen retrieval conditions18. Comparable to results found by Brinkmann et al., we found that higher incubation temperatures (>55 ˚C) resulted in optimal staining of histones in the nuclei and decondensed chromatin19. However, we found that the staining intensity of myeloperoxidase, a granular protein found in neutrophils and NETs, was low under the proposed conditions. The poor immunoreactivity of myeloperoxidase was consistent despite the use of a feline-specific antibody (Supplement Figure 1). Therefore, we encourage investigators to modify the duration and conditions (e.g., pH, temperature) of the antigen retrieval process to yield a satisfactory signal based on the antigen of interest.
One of the challenges of identifying NETs in veterinary species is the lack of species-specific antibodies. To prevent the interference encountered when using primary antibodies originating from the same species, we included an additional blocking step utilizing a high concentration of rabbit immunoglobulins to saturate any remaining binding sites on the goat anti-rabbit secondary antibodies. A major disadvantage of this technique is that it is time-consuming, because it requires multiple incubation steps. Investigators should include two different controls that exclude either primary antibody in the second immunolabeling step to ensure that the secondary antibody from either immunolabelling step binds specifically to its primary antibody. The specificity of the identified NET structures can be further verified by DNase digestion or the inclusion of biological controls consisting of aortic bifurcations from cats without CATE (Figure 5). In addition, negative controls consisting of the same concentration of isotype control antibodies as the primary antibodies should be included to rule out nonspecific antibody interactions, nonspecific binding to Fc receptors, and cellular autofluorescence. Investigators are advised to modify this protocol based on the availability of species-specific antibodies. If no species-specific antibodies are available, we advise to first evaluate the immunoreactivity of antibodies using immunocytochemistry or evaluate the protein transcript from the species of interest for homology to the referenced transcripts.
Factors like sample fixation, inadequate deparaffinization, and the presence of specific tissue components can lead to autofluorescence in thrombi. During aldehyde fixation, amines may combine with aldehydes to form Schiff base complexes, which emit fluorescence22. Incomplete deparaffinization may also chemically modify the proteins in the tissue, creating autofluorescence23. Extracellular components in vascular samples such as collagen, elastin, and red blood cells are reported to naturally fluoresce in mammals24,25. Because natural or iatrogenic autofluorescence is most noticeable in the green wavelengths (excitation = 488 nm, emission = 500–550 nm), the use of far-red fluorophores may minimize some autofluorescence26. In the present protocol, we utilized a commercially available autofluorescence quenching kit designed to electrostatically bind to autofluorescent tissue elements. We recommend that investigators optimize the duration of autofluorescence quenching, because the manufacturer’s recommendation of 5 min may diminish immunoreactivity of less abundant proteins. Alternatively, investigators can also dampen autofluorescence using Sudan Black B, 3,3’-diaminobenzidine or trypan blue27.
Because NETs are heterogeneously distributed within a thrombus, a thorough mapping of the entire aorta and iliac arteries is recommended. Regions positive for cfDNA, citH3, and NE are then magnified. The colocalization of cfDNA, citH3, and NE has been widely used to identify NET formation and to differentiate NET formation from other forms of cell death. Unlike a recent human study that found NETs to be concentrated at the periphery of coronary thrombi, most of the NETs in feline arterial thrombi were clustered at the cranial aspect of the clot28. Although we used a standardized protocol to identify NETs, microscopic evaluation and quantification of NETs remains subjective. Here, we utilized a blinded and systematic method to minimize observer bias during microscopic analysis. Because the number of NETs in a sample can be influenced by the number of neutrophils, investigators can quantify NETs relative to the number of neutrophils by identifying neutrophils based on nuclear morphology, cell diameter, and expression of neutrophil-specific proteins. Another challenge of NET identification using microscopy is that NETs have ill-defined margins and they tend to merge, forming nebulous structures. This could lead to under- or overestimation of the number of NETs in any given sample. Therefore, instead of DAPI, NETs can be stained using Sytox Green for clearer identification and denotation of cell-appendant DNA from NETs formation.
We have developed a double immunolabeling protocol to identify NETs in paraffin-embedded feline arterial thrombi. Deparaffinization, rehydration, and antigen retrieval must take place before immunolabeling of citH3 and NE. This assay can be a valuable tool for the study of NET formation in cats and provide a better understanding of the pathophysiology of CATE in cats.
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The authors have nothing to disclose.
The study was supported by funds from the University of California, Davis, Center for Companion Animal Health (CCAH 2018-30-F). The authors would like to acknowledge Dr. Kevin Woolard for usage of the fluorescence microscope.
|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|
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