Referencia

Messal, H.A., Almagro, J., Zaw Thin, M. et al. Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH. Nat Protoc 16, 239–262 (2021). https://doi.org/10.1038/s41596-020-00414-z
portada

Autores

Hendrik A. Messal, Jorge Almagro, May Zaw Thin, Antonio Tedeschi, Alessandro Ciccarelli, Laura Blackie, Kurt I. Anderson, Irene Miguel-Aliaga, Jacco van Rheenen & Axel Behrens.

Resumen

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..

Descripción

FLASH (Fast Light-microscopic analysis of Antibody-Stained wHole organs) es una nueva técnica simple, rápida y versátil de permeabilización y clarificación de tejidos para inmunofluorescencia en tres dimensiones. Mediante la utilización de un proceso de desnaturalización, solubilización de membranas y recuperación de epítopos, FLASH puede utilizarse para detectar multitud de antígenos en tejidos de ratón, biopsias de pacientes humanos, organoides y pequeños organismos como Drosophila melanogaster. FLASH permite la resolución y observación de grandes volúmenes como órganos de ratón completos, así como de estructuras subcelulares nanométricas tales como focos subnucleares o cuerpos medios durante la división celular.

Jorge Almagro

REFERENCIA DEL GRUPO INVESTIGADOR

Jorge Almagro, socio SEBBM 07420 y primer autor de este artículo junto con el Dr Hendrik Messal, es estudiante de doctorado en el laboratorio de Axel Behrens en el instituto Francis Crick (Londres, Reino Unido). El laboratorio se dedica al estudio de la heterogeneidad tumoral y las células madre del cáncer. El desarrollo de la nueva técnica de clarificación de tejidos e immunofluorescencia en 3D FLASH se ha utilizado para estudiar la arquitectura de lesiones cancerosas en pancreas (https://doi.org/10.1038/s41586-019-0891-2) y para caracterizar modelos de microcefalia en embriones de ratón (https://doi.org/10.1038/s41467-020-15359-w; https://doi.org/10.1101/2020.05.07.082032).
Síguenos en Twitter: @behrens_lab @almagro_jorge

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