Artículo del mes
febrero 2021

Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH

Referencia artículo:
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).
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.
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.
Sobre el grupo investigador