Name: Vibratome Sectioning of Agarose-Embedded Lung Tissue
Uploaded: 2023-10-06T12:50:00
Duration: 1 min 8 s
Description: Watch this Scientific Journal Video about Vibratome Sectioning of Agarose-Embedded Lung Tissue at JoVE.com

vibratome sectioning of agarose-embedded lung tissue

Innovativ lungebilleddannelse ved hjælp af agarose-indlejrede vibratomsektioner

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.

Forfatter Spotlight: Undersøgelse af rumlig proteinekspression ved hjælp af agaroseindlejrede lungevævssektioner 

Automatiseret vibratomsektionering af agaroseindlejret lungevæv til multiplexfluorescensbilleddannelse

This protocol is particularly useful in large animal models of lung transplantation and regeneration, such as pigs, where we can sample small pieces over time. This technique can help us better understand the spatial biology at play in these models. This technique produces minimally-processed lung slices suitable for immunofluorescence imaging without the need for antigen retrieval, and has been effective with all antibodies tested to date.This procedure can be adapted to cut slightly thicker sections, say around one millimeter, which can be then used in light sheet fluorescence microscopy to study the spatial protein expression in a more three-dimensional volume. Our research is investigating factors influencing lung transplant rejection, and developing therapies to reduce rejection using our pig model. This technique helps understand spatial protein changes affecting transplant outcomes post-surgery.We're currently using this technique to explore the spatial protein expression landscape in our pig lung transplant model. We hope that it will help us to better understand the mechanisms behind our experimental therapies. The key to succeeding here lies in vibratome sightings, high blade amplitudes, and lower force speeds are key things to look at if the sectioning goes badly.

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

Vibratome Sectioning of Agarose-Embedded Lung Tissue  

Vibratomsektionering af agaroseindlejret lungevæv

Til at begynde med skal du integrere lungevævet i agarose. For at forberede lungevævet til sektionering skal du først fastgøre hver vævsblok til vibratomens prøveskive med superlim og tilføje is til det omgivende bad. Derefter installeres prøveskiven forsigtigt i bufferbakken, og bakken fyldes med PBS.Indstil derefter vibratomparametrene, hold tykkelsen på 200 mikrometer, frekvensen ved 100 hertz, bladets amplitude ved 1,3 millimeter og bladets fremadgående hastighed mellem 0,02 og 0,03 millimeter pr. Sekund. Begynd sektioneringen af lungevævet. Når der er oprettet nok skiver, skal du bruge en børste til forsigtigt at løfte vævsskiven fra vibratombakken.Skiverne overføres til en plade med 24 brønde med PBS-azidopløsning. Bevar lungeskiverne ved fire grader Celsius.

Research

JoVE Journal

Medicine

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 ...