﻿autofluorescence blocking and antigen retrieval in tissue samples

Tinción e imágenes para visualizar trampas extracelulares de neutrófilos

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.

Autor destacado: Imágenes de trampas extracelulares de neutrófilos en tejidos humanos y de ratón 

Imágenes de inmunofluorescencia de trampas extracelulares de neutrófilos en tejidos humanos y de ratón

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

Autofluorescence Blocking and Antigen Retrieval in Tissue Samples  

Bloqueo de autofluorescencia y recuperación de antígenos en muestras de tejido

Para comenzar, prepare citrato 10 veces concentrado, solución de recuperación objetivo o TRS de pH seis, de acuerdo con las instrucciones del fabricante. Dilúyelo en una proporción de 1:10 con agua desionizada. Luego caliente la solución en un frasco de tinción de plástico a 96 grados centígrados.Retire los portaobjetos de la rejilla de portaobjetos y colóquelos en una cámara húmeda. A continuación, pipetee una o dos gotas del agente reductor de autofluorescencia en cada muestra de tejido e incube las muestras durante cinco minutos. Vuelva a apilar los portaobjetos en la rejilla de portaobjetos y enjuague durante un minuto en etanol al 60 % con movimientos hacia arriba y hacia abajo para eliminar el reactivo reductor de autofluorescencia no utilizado.Sumerja la rejilla deslizante en agua desionizada durante cinco minutos. Transfiera los portaobjetos a un frasco de tinción con tampón TRS precalentado para la recuperación de antígenos. Incubar los portaobjetos durante 10 minutos a 96 grados centígrados en un baño de agua.Retire el frasco de tinción y déjelo enfriar a temperatura ambiente durante aproximadamente 60 a 90 minutos. A continuación, retire el TRS mientras mantiene los portaobjetos en el frasco. Enjuague los portaobjetos dos veces con solución salina tamponada TRIS que contiene 0.05% Tween a pH 7.4 durante tres minutos cada una para eliminar cualquier residuo restante de TRS.Coloque los portaobjetos en una cámara húmeda. Limpie suavemente el exceso de agua de los portaobjetos con pañuelos de papel, luego rodee cada muestra con un bolígrafo de barrera hidrofóbica para evitar que la solución de anticuerpos se escurra.

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Research

JoVE Journal

Immunology and Infection

﻿Autofluorescence Blocking and Antigen Retrieval in Tissue Samples

291 vistas. Para comenzar, prepare citrato 10 veces concentrado, solución de recuperación objetivo o TRS de pH seis, de acuerdo con las instrucciones del fabricante. Dilúyelo en una proporción de 1:10 con agua desionizada. Luego caliente la solución en un frasco de tinción de plástico a 96 grados centígrados.Retire los portaobjetos de la rejilla de portaobjetos y colóquelos en una cámara húmeda. A continuación, pipetee una o dos gotas del agente reductor de autofluorescencia en cada mu...

Video: Autofluorescence Blocking and Antigen Retrieval in Tissue Samples  

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

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 ...