vibratome sectioning of agarose-embedded lung tissue

Imagens pulmonares inovadoras usando seções de vibratomo embebidas em agarose

Ex vivo lung tissue slices are extensively used in the study of lung disease1. Current gold standards, particularly in clinical and translational large animal studies, employ hematoxylin and eosin staining coupled with brightfield microscopy and observer-based scoring to assess and grade lung dysfunction2,3. While still a valuable technique, it exhibits limitations in terms of spatial resolution and the number of markers that can be imaged simultaneously. Moreover, lung tissue is required to undergo extensive solvent-based washes, which results in dehydration, shrinkage, and rehydration of the tissue before its final imaging4. This process is not only time-consuming but is also environmentally unfriendly, masks protein epitopes, and may invoke structural tissue changes5,6,7,8. However, for lung tissue, embedding in paraffin prior to sectioning has been a necessity owing to the structural fragility of the lung. In contrast, solid organs such as the brain can be cut using a vibrating microtome (vibratome), both in fixed and fresh tissue9,10,11,12.
To enable vibratome sectioning in lung tissue, a method termed precision-cut lung slices&#160;(PCLS) was established where low melting point agarose is injected into fresh lung tissue via the airways or blood vessels and left to solidify13,14. The agarose in the airways provides sufficient structural support to then allow vibratome sectioning9,14. In rodents, this is simple to achieve as agarose can be injected via the trachea; however, in pig and human tissue, it can be challenging to locate a suitable airway/vessel within a given biopsy15,16. Moreover, even if such an airway/vessel is located, substantial force is usually required to inject the agarose, which may influence subsequent lung morphology17.
To overcome the challenges experienced in paraffin embedding/sectioning/staining and the PCLS workflow, we have established a new processing pipeline for fixed lung tissue. This workflow allows the use of a vibratome for sectioning, can produce thinner slices than in PCLS, and yields slices that do not require antigen retrieval for multiplex fluorescence imaging. This method offers a means of &#34;least processing&#34; to generate lung sections that can be used in advanced light microscopy. Moreover, imaging readouts can be used to demonstrate the spatial relationships of cells and anatomical structures within the lung tissue by capturing the volumetric architecture of the tissue18,19,20,21. This paper describes the protocol to generate these lung sections and demonstrates multiplex stains applied to each of these tissues and how these advanced imaging data can be used for quantification.

Autor em destaque: Estudando a expressão espacial de proteínas usando seções de tecido pulmonar embebidas em agarose 

Secção Automatizada por Vibratomo de Tecido Pulmonar Incorporado em Agarose para Imagem por Fluorescência Multiplex

Watch this Scientific Journal Video about Vibratome Sectioning of Agarose-Embedded Lung Tissue at JoVE.com

Vibratome Sectioning of Agarose-Embedded Lung Tissue  

Secção em Vibratomo de Tecido Pulmonar Embutido em Agarose

Para começar, incorpore o tecido pulmonar em agarose. Para preparar o tecido pulmonar para secção, primeiro, prenda cada bloco de tecido ao disco da amostra do vibratomo com super cola e adicione gelo ao banho circundante. Em seguida, instale cuidadosamente o disco da amostra na bandeja tampão e encha a bandeja com PBS.Em seguida, defina os parâmetros do vibratom, mantendo a espessura em 200 micrômetros, a frequência em 100 hertz, a amplitude da lâmina em 1,3 milímetros e a velocidade de avanço da lâmina entre 0,02 e 0,03 milímetros por segundo. Iniciar a secção do tecido pulmonar. Uma vez que fatias suficientes tenham sido criadas, use um pincel para levantar suavemente a fatia de tecido da bandeja de vibratomo.Transfira as fatias para uma placa de 24 poços com solução de azida PBS. Preservar as fatias de pulmão a quatro graus Celsius.

vibratome-sectioning-of-agarose-embedded-lung-tissue

Research

JoVE Journal

Medicine

Vibratome Sectioning of Agarose-Embedded Lung Tissue

1.0K visualizações. Para começar, incorpore o tecido pulmonar em agarose. Para preparar o tecido pulmonar para secção, primeiro, prenda cada bloco de tecido ao disco da amostra do vibratomo com super cola e adicione gelo ao banho circundante. Em seguida, instale cuidadosamente o disco da amostra na bandeja tampão e encha a bandeja com PBS.Em seguida, defina os parâmetros do vibratom, mantendo a espessura em 200 micrômetros, a frequência em 100 hertz, a amplitude da lâmina em 1,3 milímetros e a ve...

Vídeo: Vibratome Sectioning of Agarose-Embedded Lung Tissue  

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...