﻿autofluorescence blocking and antigen retrieval in tissue samples

Nötrofil hücre dışı tuzakları görselleştirmek için boyama ve görüntüleme

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.

Yazar Spotlight: İnsan ve Fare Dokularında Nötrofil Hücre Dışı Tuzaklar Görüntüleme 

İnsan ve Fare Dokularında Nötrofil Hücre Dışı Tuzakların İmmünofloresan Görüntülemesi

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  

Doku Örneklerinde Otofloresan Blokajı ve Antijen Alımı

Başlamak için, üreticinin talimatlarına göre 10 kat konsantre sitrat, hedef alma çözeltisi veya pH altı TRS hazırlayın. Deiyonize su ile 1:10 oranında seyreltin. Daha sonra çözeltiyi plastik bir boyama kavanozunda 96 santigrat dereceye ısıtın.Slaytları slayt rafından çıkarın ve ıslak bir hazneye yerleştirin. Daha sonra, her bir doku örneğine bir veya iki damla otofloresan azaltıcı ajan pipetleyin ve örnekleri beş dakika inkübe edin. Slaytları tekrar sürgü rafına yerleştirin ve kullanılmayan otofloresan azaltıcı reaktifi çıkarmak için yukarı ve aşağı hareketler kullanarak %60 etanol içinde bir dakika durulayın.Sürgülü rafı beş dakika boyunca deiyonize suya batırın. Slaytları, Antijen Alımı için önceden ısıtılmış TRS Tamponu ile bir boyama kavanozuna aktarın. Slaytları bir su banyosunda 96 santigrat derecede 10 dakika inkübe edin.Boyama kavanozunu çıkarın ve yaklaşık 60 ila 90 dakika oda sıcaklığına soğumaya bırakın. Ardından, slaytları kavanozda tutarken TRS'yi çıkarın. Kalan TRS kalıntılarını gidermek için slaytları pH 7.4'te %0.05 Tween içeren TRIS Tamponlu Salin ile her biri üç dakika boyunca iki kez durulayın.Slaytları ıslak bir odaya yerleştirin. Kağıt mendiller kullanarak slaytlardaki fazla suyu nazikçe silin, ardından antikor solüsyonunun akmasını önlemek için her numuneyi hidrofobik bir bariyer kalemi kullanarak daire içine alın.

Research

JoVE Journal

Immunology and Infection

﻿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 ...