Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH.
Nat Protoc
; 16(1): 239-262, 2021 01.
Article
en En
| MEDLINE
| ID: mdl-33247285
ABSTRACT
Advances in light-sheet and confocal microscopy now allow imaging of cleared large biological tissue samples and enable the 3D appreciation of cell and protein localization in their native organ environment. However, the sample preparations for such imaging are often onerous, and their capability for antigen detection is limited. Here, we describe FLASH (fast light-microscopic analysis of antibody-stained whole organs), a simple, rapid, fully customizable technique for molecular phenotyping of intact tissue volumes. FLASH utilizes non-degradative epitope recovery and membrane solubilization to enable the detection of a multitude of membranous, cytoplasmic and nuclear antigens in whole mouse organs and embryos, human biopsies, organoids and Drosophila. Retrieval and immunolabeling of epithelial markers, an obstacle for previous clearing techniques, can be achieved with FLASH. Upon volumetric imaging, FLASH-processed samples preserve their architecture and integrity and can be paraffin-embedded for subsequent histopathological analysis. The technique can be performed by scientists trained in light microscopy and yields results in <1 week.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Técnica del Anticuerpo Fluorescente
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Microscopía Confocal
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Imagenología Tridimensional
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Antígenos
Límite:
Animals
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Female
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Humans
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Male
Idioma:
En
Revista:
Nat Protoc
Año:
2021
Tipo del documento:
Article
País de afiliación:
Reino Unido