Reference

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

Authors

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.

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..

Description

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

Leer más

Did you publish an interesting article recently?

Send it through our application form and we will contact you. Age limit: 32.

The selected articles will participate at the Award to the best article of young people of the SEBBM which will be given during SEBBM conference, that will take place at Spain (free registration, travel and accommodation).

More articles of the month

Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH

25-01-2021

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...

Read more

Viral Bcl2s' transmembrane domain interact with host Bcl2 proteins to control cellular apoptosis

08-01-2021

Viral control of programmed cell death relies in part on the expression of viral analogs of the B-cell lymphoma 2 (Bcl2) protein known as viral Bcl2s (vBcl2s). vBcl2s control apoptosis...

Read more

Macrophages promote endothelial-to-mesenchymal transition via MT1-MMP/TGFß after myocardial infarction

01-12-2020

Macrophages (Mφs) produce factors that participate in cardiac repair and remodeling after myocardial infarction (MI); however, how these factors crosstalk with other cell types mediating repair is not fully understood...

Read more

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics

30-10-2020

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic...

Read more

Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas

01-10-2020

C3G is a guanine nucleotide exchange factor (GEF) that regulates cell adhesion and migration by activating the GTPase Rap1. The GEF activity of C3G is stimulated by the adaptor proteins...

Read more

Expression of the long non-coding RNA TCL6 is associated with clinical outcome in pediatric B-cell acute lymphoblastic leukemia

31-08-2020

The reciprocal translocation t(12;21)(p13;q22)[ETV6/RUNX1] is the most frequent chromosomal rearrangement in pediatric B-cell acute lymphoblastic leukemia(B-ALL). Long non-coding RNAs (lncRNAs) play important roles in numerous diseases and they represent an...

Read more

Evaluation of different approaches used to study membrane permeabilization by actinoporins on model lipid vesicles

30-07-2020

Release of aqueous contents from model lipid vesicles has been a standard procedure to evaluate pore formation efficiency by actinoporins, such as sticholysin II (StnII), for the last few decades...

Read more

ADAR1-mediated RNA editing is a novel oncogenic process in thyroid cancer and regulates miR-200 activity

01-07-2020

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. A-to-I editing of RNA is a widespread posttranscriptional process that has recently emerged as an important mechanism...

Read more

Sarcoplasmic reticulum Ca2+ decreases with age and correlates with the decline in muscle function in Drosophila

29-05-2020

Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown...

Read more

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

30-04-2020

Core-fucosylation is an essential biological modification by which a fucose is transferred from GDP-β-L-fucose to the innermost N-acetylglucosamine residue of N-linked glycans. A single human enzyme α1,6-fucosyltransferase (FUT8) is the...

Read more

Protector Members