agarose embedding of lung tissue for multiplex fluorescence imaging

アガロース包埋ビブラトーム切片を用いた革新的な肺イメージング

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.

著者スポットライト:アガロース包埋肺組織切片を用いた空間的タンパク質発現の研究 

マルチプレックス蛍光イメージングのためのアガロース包埋肺組織の自動ビブラトーム切片化

Watch this Scientific Journal Video about Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging at JoVE.com

Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging  

マルチプレックス蛍光イメージングのための肺組織のアガロース包埋

まず、ブタの肺組織を室温で4%パラホルムアルデヒドで48時間固定します。次に、0.01%アジ化ナトリウムを含む30ミリリットルのPBSを含む50ミリリットルの円錐形チューブに肺組織を移します。プラスチックカップ、細かい鉗子、滅菌ハサミを用意しておいてください。アガロースを沸点に達するまで電子レンジで加熱します。その後、水浴で摂氏42度まで冷やします。使用する準備ができるまで、ウォーターバスに保管してください。PBSアジド溶液から肺生検を除去します。細かい鉗子と滅菌ハサミを使用して、肺葉を慎重に小さなブロックに解剖します。次に、プラスチックカップの真ん中を切り取ります。カップの底に少量のアガロースを注ぎます。50ミリリットルの円錐形チューブの蓋を、縁を外してアガロの表面に置きます。このセットアップを摂氏4度で5分間保存し、アガロースが固まるようにします。アガロースが固まったら、プラスチックカップを冷蔵庫から取り出します。次に、約1ミリリットルの液体アガロースを蓋に注ぎます。次に、肺組織ブロックを蓋の上にそっと置きます。アガロースが半固化したら、肺組織を液体アガロースにゆっくりと浸します。アガロースが固まるまで、摂氏4度で約15分間冷蔵します。鋭利な刃を使用して、肺組織を取り巻く余分なアガロースを慎重に取り除きます。.

agarose-embedding-of-lung-tissue-for-multiplex-fluorescence-imaging

Research

JoVE Journal

Medicine

Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging

520 ビュー. まず、ブタの肺組織を室温で4%パラホルムアルデヒドで48時間固定します。次に、0.01%アジ化ナトリウムを含む30ミリリットルのPBSを含む50ミリリットルの円錐形チューブに肺組織を移します。プラスチックカップ、細かい鉗子、滅菌ハサミを用意しておいてください。アガロースを沸点に達するまで電子レンジで加熱します。その後、水浴で摂氏42度まで冷やします?...

ビデオ: Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging  

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 ...