Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging
(2023) In Journal of Visualized Experiments 2023(200).- Abstract
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.... (More)
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 µm to 70 µm 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.
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- author
- Wang, Qi LU ; Bechet, Nicholas B. LU and Lindstedt, Sandra LU
- organization
-
- Thoracic Surgery
- StemTherapy: National Initiative on Stem Cells for Regenerative Therapy
- WCMM-Wallenberg Centre for Molecular Medicine
- MultiPark: Multidisciplinary research focused on Parkinson´s disease
- LUCC: Lund University Cancer Centre
- Clinical and experimental lung transplantation (research group)
- DCD transplantation of lungs (research group)
- NPWT technology (research group)
- publishing date
- 2023-10
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Visualized Experiments
- volume
- 2023
- issue
- 200
- article number
- e65943
- publisher
- JoVE
- external identifiers
-
- pmid:37870323
- scopus:85174706039
- ISSN
- 1940-087X
- DOI
- 10.3791/65943
- language
- English
- LU publication?
- yes
- id
- c08bc9c0-dba6-4056-94e5-ccc43c0e0301
- date added to LUP
- 2023-12-11 14:06:54
- date last changed
- 2024-09-11 21:04:05
@article{c08bc9c0-dba6-4056-94e5-ccc43c0e0301, abstract = {{<p>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 µm to 70 µm 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.</p>}}, author = {{Wang, Qi and Bechet, Nicholas B. and Lindstedt, Sandra}}, issn = {{1940-087X}}, language = {{eng}}, number = {{200}}, publisher = {{JoVE}}, series = {{Journal of Visualized Experiments}}, title = {{Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging}}, url = {{http://dx.doi.org/10.3791/65943}}, doi = {{10.3791/65943}}, volume = {{2023}}, year = {{2023}}, }