耳部炎症和耳部整体染色

Jia Tong Loh; Merry Gunawan; I-hsin Su

doi:10.21769/BioProtoc.1967

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Peer-reviewed

Ear Inflammation and Whole-mount Ear Staining

耳部炎症和耳部整体染色

Jia Tong Loh

MG Merry Gunawan

I-hsin Su

发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1967 浏览次数: 11490

评审: Ivan ZanoniGriselda Zuccarino-CataniaBenoit Stijlemans

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The authors used this protocol in:

Cover of Nature Immunology, featuring study using the protocol.

Apr 2015

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Abstract

The recruitment of circulating neutrophils from the bloodstream to the site of inflammation represents one of the earliest events during an innate immune response. During this response, neutrophils tether and roll along the vessel walls before transmigrating across the endothelium into the interstitial space to exert their functions. Here, we describe a protocol for the staining of intravascular and tissue-localized neutrophils following contact sensitization of the skin with croton oil. Visualization of the neutrophilic distribution in skin provides for a better interpretation of the local immune response.

Keywords: Neutrophils (中性白细胞)

Ear inflammation (耳朵发炎)

Skin inflammation (皮肤炎症)

Background

Characterisation of neutrophil distribution within the skin following inflammation represents an important avenue for the understanding of their specialized functions. Even though the recruitment of neutrophils to the inflamed skin has been widely characterized by flow cytometry (Hampton et al., 2015; Stock et al., 2014), such technique provides limited insight on the intravascular versus interstitial localization of neutrophils. Recirculating and tissue-localized neutrophils exhibit different phenotypes and functions, which necessitates their discrimination to identify key players of the local immune response. With the development of intravital microscopy (IVM), the direct visualization of fluorescently tagged immune cells in vivo is made possible. However, due to the high cost of IVM, the accessibility of this powerful imaging tool to researchers is limited. Here, we present an alternative protocol for the high resolution static imaging of neutrophils in skin by making use of cost-effective reagents and commonly available confocal laser-scanning microscopy.

Materials and Reagents

Cotton swabs
Sterilin^TM Petri dish (90 mm) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 101IRR )
24-well cell culture plates (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 142475 )
Glass slides (Biomedical Sciences Institutes, catalog number: BMH.880103 )
Fisherbrand^TM cover glass (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-545-81 )
Clear nail polish
Mice
Croton oil (Sigma-Aldrich, catalog number: C6719 )
Veet hair removal cream
Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
Acetone (Thermo Fisher Scientific, Fisher Scientific, catalog number: 67-64-1 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 10735108001 )
Sodium azide (Sigma-Aldrich, catalog number: S2002 )
Antibodies

PE anti-mouse Ly-6G antibody (clone 1A8) (BioLegend, catalog number: 127607 )
PE rat IgG2a, κ Isotype control antibody (clone RTK2758) (BioLegend, catalog number: 400507 )
APC anti-mouse CD31 (clone MEC 13.3) (BD, Pharmingen^TM, catalog number: 561814 )