﻿autofluorescence blocking and antigen retrieval in tissue samples

Staining and Imaging to Visualize Neutrophil Extracellular Traps

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 presence of these proteins; indeed, it has the advantage of specifically detecting NET-like structures. In the NETs, the mentioned proteins co-localize with extracellular DNA, which can be detected by an overlap of the fluorescence signals of each stained protein and the extracellular DNA. In contrast to the overlapping signals due to extracellular DNA and protein co-localization in NETs, intact neutrophils show no co-localization. Here, the NET components are usually stored separately in the granules, nuclei, and cytosol3.
Since their first discovery, it has been shown that NETs play a central role in numerous diseases, particularly those involving inflammation. NETs show antimicrobial functions during infection through trapping and killing extracellular pathogens in blood and tissue4,5. However, NETs have also been connected to autoimmune diseases and hyperinflammatory responses, like systemic lupus erythematosus, rheumatic arthritis, and allergic asthma6,7,8. NETs promote vaso-occlusion and inflammation in atherosclerosis, platelet adhesion, and are speculated to play a role in metastatic cancer9,10,11. Nevertheless, they are thought to have anti-inflammatory properties by reducing proinflammatory cytokine levels12. While NETs are gaining more interest in a broader field of research, a robust NET detection method is fundamental for future research.
Even though the visualization of NETs in different tissue using immunofluorescence imaging is complex and requires customization, apart from electron microscopy, it is currently one of the most renowned methods for visualizing the interactions between NETs and cells and is predominantly used in formalin-fixed paraffin-embedded tissues (FFPE)13,14. However, comparing NET imaging is difficult, as different laboratories use their own customized protocols. These protocols differ in their use of antibodies, antigen retrieval, or permeabilization method and are often optimized for a specific type of tissue3,13,15,16,17,18,19,20,21,22,23,24,25,26,27.
After Brinkmann et al. published the first methodic study using immunofluorescent visualization of NETs in FFPE tissue, we wanted to optimize this protocol for a wider variety of tissues and species15. Additionally, to establish a broadly applicable immunofluorescence protocol, we tested different modified protocols from studies that used immunofluorescence methods in FFPE tissue to detect NETs3,13,16,17,18,19,20,21,22,23,24,25,26,27. Furthermore, we tried a new H3cit antibody for more specific extracellular staining28. We hypothesize that by systematically adapting current staining protocols to different species and tissue, in vitro imaging can be improved, resulting in a better representation of the interaction between neutrophils and NETs both locally and systemically.

Author Spotlight: Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues 

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Among antibody selection for tissue imaging, the fixation process poses a challenge by epitope masking. Therefore, customized retriever step is required to expose these antigens for antibody binding. Additionally, tissue inherent autofluorescence can cause difficulties through to a high background staining.However, the use of different approaches in the protocols in the literature hinders a standardized comparison between the images. Our study aims to address the demand for standardized procedures and assist the researcher in overcoming challenges during the imaging process. We established a versatile imaging protocol applicable to different tissues by comparing various different protocols.So our work provides guiding and troubleshooting tips from antibody usage to sample mounting, highlighting the potential pitfalls. Our findings advance research by introducing a new primary antibody for citrullinated hisone three, and an autofluorescent reducing agent to minimize background staining. Furthermore, for successful imaging, we emphasize the careful handling of samples and the importance of maintaining a constant high temperature for antigen retrieval.

Watch this Scientific Journal Video about ﻿Autofluorescence Blocking and Antigen Retrieval in Tissue Samples at JoVE.com

Autofluorescence Blocking and Antigen Retrieval in Tissue Samples  

﻿Autofluorescence Blocking and Antigen Retrieval in Tissue Samples

To begin, prepare 10 times concentrated citrate, target retrieval solution or TRS of pH six, according to the manufacturer's instructions. Dilute it to a ratio of 1:10 with deionized water. Then heat the solution in a plastic staining jar to 96 degrees Celsius.Remove the slides from the slide rack and place them into a wet chamber. Next, pipette one or two drops of the autofluorescence-reducing agent onto each tissue sample and incubate the samples for five minutes. Stack the slides back into the slide rack, and rinse for one minute in 60%ethanol using upward and downward motions to remove the unused autofluorescence-reducing reagent.Submerge the slide rack in deionized water for five minutes. Transfer the slides into a staining jar with preheated TRS Buffer for Antigen Retrieval. Incubate the slides for 10 minutes at 96 degrees Celsius in a water bath.Remove the staining jar and let it cool to room temperature for approximately 60 to 90 minutes. Next, remove the TRS while keeping the slides in the jar. Rinse the slides twice with TRIS Buffered Saline containing 0.05%Tween at pH 7.4 for three minutes each to remove any remaining TRS residues.Place the slides in a wet chamber. Gently wipe excess water from the slides using paper tissues, then encircle each sample using a hydrophobic barrier pen to prevent the antibody solution from running off.

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Research

JoVE Journal

Immunology and Infection

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 &#181;m 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.

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.

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...

Immunofluorescence Staining of Neutrophil Extracellular Traps in Tissue Samples

Begin by pipetting one drop of ready-to-use blocking solution with donkey serum onto the tissue samples. Then incubate the samples for 30 minutes at room temperature. Remove the excess blocking solution from the slides by tapping the edge against a hard surface.Dilute the primary antibodies in the antibody dilution buffer to prepare 100 microliters of the final solution for each sample. Set up the antibodies, one tube for each primary antibody and one for each isocontrol antibody. Evenly spread 100 microliters of isocontrol antibodies to one sample, and 100 microliters of myeloperoxidase and citrullinated histone H3, or myeloperoxidase and neutrophil elastase antibody dilution to the other sample.Place the slides in a wet chamber and store them overnight at four degrees Celsius. The following day, tap off excess primary antibody solution from the slides and stack them in a cuvette. Rinse the slides three times for five minutes each using Tris buffered saline with tween To remove the remaining primary antibody solution.Prepare two distinctly fluorescent secondary antibodies, donkey anti-goat for myeloperoxidase and donkey anti-rabbit for citrullinated histone H3 staining. Dilute each antibody to 7.5 micrograms per milliliter concentration in the antibody dilution buffer. Place the slides in a wet chamber and dispense 100 microliters of the secondary antibody solution onto each sample.Incubate them for 30 minutes at room temperature protected from light. Remove excess secondary antibody solution by tapping the slides. Put the slides in a slide rack.Submerge the slides three times for five minutes each in a staining jar filled with PBS to wash off unbound antibodies. Prepare the dappy solution and dilute it to a concentration of one microgram per milliliter with deionized water. Submerge the slide rack in a staining jar containing the dappy solution and incubate for five minutes at room temperature in the dark.Next, submerge the slide rack in a staining jar with PBS for five minutes to wash off any excess dappy solution. Finally, mount the samples using cover slips and mounting medium. The citrullinated histone H3 antibody only bound to the extracellular histones and showed no staining of intracellular histones.The human or mouse specific myeloperoxidase antibody showed no specific staining. While the universal human and mouse antibodies showed consistent good staining for myeloperoxidase. When no blocking agent was used, some mouse samples showed more non-specific and partial positive staining.When blocked, the five minutes blocking time showed good results compared to the 10 minutes blocking time. The higher temperatures in the microwave and water bath showed consistently moderate to good antigen retrieval. Whereas in a 60 degrees Celsius water bath there was only partially positive to no staining.For double staining, more favorable results were obtained for the samples incubated at 96 degrees Celsius water bath. However, exceeding the incubation time of 40 minutes at 96 degrees Celsius resulted in less intense antibody staining.