The authors gratefully acknowledge funding received from the Wallenberg Molecular Medicine Foundation and Stem Cell Center of Lund University and acknowledge the Lund University Bioimaging Centre (LBIC) for access to the Nikon A1RHD.

Mouse lungs were donated by researchers using other organ systems in an effort to uphold the 3Rs. All procedures in pigs were carried out in accordance with the European directive 2010/63/EU and were approved by Malm&#246;-Lund Ethical Committee on Animal Research (Dnr 5.8.18-05527/2019) and conducted according to the CODEX guidelines of the Swedish Research Council. Approval for the use of human samples was granted by the Swedish National Ethics Committee (Dnr 2020-07115 and Dnr 2020-01864). See the Table of Materials for details related to all materials and instruments used in this protocol.
1. Preparation of solutions and materials
<ol>
	<li>Fix the lung tissue in 4% paraformaldehyde at room temperature for 48 h. Subsequently, transfer the lung tissue to a 50 mL conical tube filled with Phosphate-buffered saline (PBS) containing 0.01% sodium azide.</li>
	<li>Prepare a 3% (w/v) agarose solution by dissolving 1.5 g of low-melting-point agarose in 50 mL of sterile PBS. Ready a plastic cup, fine forceps, and sterile scissors.</li>
	<li>Heat the agarose in a microwave until it boils, then cool it down to 42 &#176;C in a water bath. Keep the agarose in its liquid form by storing it in the water bath until ready to use. 
	NOTE: While heating the agarose, watch the microwave until it begins to boil so it does not overflow. This usually takes around 1.5 min.</li>
</ol>
2. Lung tissue embedded in agarose
<ol>
	<li>Lift the lung biopsy from the PBS-azide and carefully dissect the lung lobes using fine forceps and sterile scissors. Cut the lung lobe into smaller blocks. 
	NOTE: While sectioning with pleura is possible, its thick elastin bundles can impede cutting with the vibratome blade. Thus, we recommend its removal if it is not necessary.</li>
	<li>Cut a plastic cup through the middle and add a small amount of agarose to its bottom. Place the lid from a 50 mL conical tube (rim removed) on the surface of the agarose and store the cup at 4 &#176;C for 5 min to allow the agarose to solidify for later use.</li>
	<li>Retrieve the plastic cup from the 4 &#176;C refrigerator, add approximately 1 mL of liquid agarose onto the lid, then gently place the lung tissue block onto the lid. Leave the agarose for 2-3 min to semi-solidify at room temperature. Then, slowly pour the liquid agarose onto the lung tissue until it is fully submerged.</li>
	<li>Refrigerate at 4 &#176;C for approximately 15 min until the agarose solidifies. Next, carefully use a sharp blade to remove extra agarose surrounding the lung tissue, leaving a uniform layer of approximately 5 mm thickness around the tissue (Figure 1).</li>
</ol>
3. Cutting lung tissue sections
<ol>
	<li>To prepare the lung tissue for sectioning, attach each tissue block onto the specimen disk of the vibratome using super glue. Next, fill the vibratome buffer tray with PBS and place ice in the surrounding ice bath. Finally, carefully install the specimen disk into the buffer tray.</li>
	<li>Slice the lung tissue with the vibratome with the following settings: thickness: 200 &#181;m, frequency: 100 Hz, amplitude of the blade: 1.3 mm, and forward speed of the blade of 0.02-0.03 mm/s, which depends on tissue stiffness. 
	NOTE: Cutting is possible down to a thickness of 70 &#181;m if the blade speed is reduced to 0.01 mm/s. Certain vibratomes (e.g., Leica V1200s) allow automation of sectioning by setting a cutting window.</li>
	<li>To collect sections, gently transfer the tissue slice by scooping it with a brush from the vibratome tray into a 24-well plate filled with 0.01% azide in PBS. Finally, preserve the lung slices at 4 &#176;C.</li>
</ol>
4. Immunostaining of elastin, CD31, HTII-280, and SMA
<ol>
	<li>Permeabilize and block (1% bovine serum albumin + 0.5% Triton + 5% normal goat serum) the tissue at 4 &#176;C under gentle shaking for 45 min.</li>
	<li>Dilute the primary antibodies&#160;(SMA 1:500; elastin 1:250; CD31 1:250; HTII-280 1:250) in PBS, add 300 &#181;L in per well, and incubate overnight at 4 &#176;C under gentle shaking.</li>
	<li>Without touching the slice and removing the primary antibodies with a pipette tip, wash 3 x 20 min (400 &#181;L) with PBS.</li>
	<li>Dilute the secondary antibodies&#160;(1:1000) in PBS, add 300 &#181;L in per well, and incubate at 4 &#176;C under gentle shaking for 90 min.</li>
	<li>Remove the secondary antibodies with a pipette tip, add 4&#8217;,6-diamidino-2-phenylindole (DAPI)(1:1000) and tomato lectin-488 (1:500) for a 30 min incubation, and wash for 3 x 10 min with PBS.</li>
	<li>Place three drops of mounting medium on the slide, mount a coverslip, and proceed to imaging.</li>
</ol>

Innovative Lung Imaging Using Agarose-Embedded Vibratome Sections

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The authors have no conflicts of interest to disclose.

In this protocol, we present an improved vibratome-based method for generating lung sections. Compared to paraffin-based processing techniques, this method is more cost effective, time efficient, and better for the environment22. Moreover, this method helps maintain the structural integrity of pulmonary tissue slices and allows for advanced immunofluorescence imaging without the need for antigen retrieval. However, during our experiments, we also found certain limitations to this workflow. Diseased lungs can interfere with the cutting process due to tissue destruction caused by pathological changes. While using the vibratome for sectioning, airway obstructions and severe fibrotic lung tissue can impede the blade, making the process of slicing these tissues more challenging. However, this can be overcome by reducing blade speed and supporting the tissue with a fine paintbrush. Similar challenges arise from the thickening of the pleura, which is a common outcome in pleurisy, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF)23,24,25. If the pleura is not of interest for an experiment, we recommend removing it before sectioning or by isolation a lung volume away from the pleura. If the pleura is necessary, then it can be sliced using a similar manipulation as above, involving slower blade speeds and slice support from a fine brush.
While precision-cut lung slices can preserve the three-dimensional architecture and native environment of the lung26, it is important to note that generating PCLS is a complex and time-consuming process. The success of generating lung tissue slices greatly depends on the efficiency of agarose filling, which, in turn, is influenced by the presence of intact pleura and viable injection sites in the obtained tissue. This is simple to achieve in rodents as agarose can easily be introduced through the trachea into intact lungs27,28,29. However, in large animal research, biopsies taken across an experiment are typically from distal lung segments, which lack a large enough airway to cannulate30,31. Thus, instead of injecting agarose into the bronchus or blood vessels, we adopt this method where lung tissue, regardless of species of origin or sample volume, is embedded in low-melting-point agarose to generate lung tissue slices. This approach is technically easier and aims to minimize tissue damage as much as possible. Based on our experience, this method has demonstrated higher efficiency and ease in generating lung tissue sections. It effectively preserves the morphology of alveoli and is particularly suitable for cutting lung tissue, enabling the generation of repeatable high-resolution multiplex fluorescence imaging.
In this study, we further demonstrate two sample-based quantifications for the imaging data generated. The first utilized image tracing to assess differences in alveolar size across species samples. Unsurprisingly, mouse alveoli were the smallest, while pig and human alveoli were similar in size, highlighting pigs as an interesting translational model for lung research.
For the second quantification, we compared HTII distribution between an adult and infant lung sample. Type 2 pneumocytes play a vital role in the structure of the distal lung epithelium. This unique capability of TIIs contributes to the repair and regeneration of the alveolar epithelium32. In the healthy adult human lung, TIIs account for approximately 15% of the total cell population, while their coverage of the alveolar surface area is estimated to be around 5%33. HTII-280 is a widely recognized lung-specific marker for studying the development and response of alveolar epithelial cells to injury, and it is specifically localized to the apical plasma membranes of TIIs. In the experiment, adult tissue was obtained from a donor patient who died due to intracerebral hemorrhage and had concurrent lung injury, while the infant sample came from an asphyxiation-related mortality. Our findings indicate that the expression of HTII-280 was significantly lower in the adult lung sample than in the infant sample. Interestingly, while HTII-280 is expressed in the majority of HTIIs, its expression can also be influenced by tissue quality, and it is significantly reduced in injured human lung tissue34. Aged lung tissues exhibit a significant decrease in the number and secretory activity of TIIs compared to young tissues, and elderly individuals demonstrate considerably lower proliferation, secretion, and anti-apoptotic activity of TIIs in comparison to those young tissues35. In keeping with this, the identification and detection of a cell-surface protein specific to human TIIs could aid in assessing the severity of lung injury and evaluating treatment approaches aimed at enhancing lung repair36,37. Thus, utilizing this pipeline, it would be of great interest to conduct alveolar TII studies in larger human cohorts of health and disease.
In conclusion, this protocol presents a faster and easier approach for cutting lung tissue. This method provides detailed instructions for lung tissue preparation, cutting, and staining, ensuring the preservation of the intact lung tissue structure. It optimizes the model for studying both healthy and diseased lung architecture, leading to improved experimental efficiency. Overall, this study introduces a promising approach to investigate complex spatial biology in lung tissue pathophysiology across species, which can help to better understand the molecular mechanisms at play in disease and repair.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 well tissue culture inserts</td>
			<td>SARSTEDT</td>
			<td>83.3932.300</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>SOLVECO</td>
			<td>6095714</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;, 6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>&#160;SARSTEDT</td>
			<td>2045221</td>
			<td></td>
		</tr>
		<tr>
			<td>Cluster of differentiation 31 (CD31)</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Nikon</td>
			<td>A1R HD25</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastin</td>
			<td>Abcam</td>
			<td>ab23747</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia X1000 Coverslip</td>
			<td>Thermo Fisher scientific</td>
			<td>10318963</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia SuperFrost Plus Adhesion slides</td>
			<td>Thermo Fisher scientific</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>AESCULAP</td>
			<td>FB395R</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse 647&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>A21235</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit 568</td>
			<td>Invitrogen&#160;</td>
			<td>A11011</td>
			<td></td>
		</tr>
		<tr>
			<td>Human type II cells</td>
			<td>Terrace Biotech</td>
			<td>TB-27AHT2-280&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Fluoromount-G Mounting Medium</td>
			<td>Thermo Fisher scientific</td>
			<td>E41473</td>
			<td></td>
		</tr>
		<tr>
			<td>Low gelling temperature Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>1003467046</td>
			<td></td>
		</tr>
		<tr>
			<td>Lycopersicon Esculentum lectin</td>
			<td>Thermo Fisher scientific</td>
			<td>2531965</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher scientific</td>
			<td>50-100-8798</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cup</td>
			<td>kontorsgiganten</td>
			<td>885221</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>STILLE</td>
			<td>101-8380-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Smooth muscle actin</td>
			<td>Abcam</td>
			<td>ab5694</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>K54329188239</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>LOCTITE</td>
			<td>2721643</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The process of generating lung tissue slices involves several key steps, including preparation, embedding, and sectioning. The agarose solution (3% (w/v) is made by dissolving 1.5 g of low-melting-point agarose in 50 mL of sterile PBS. Consumables needed include a plastic cup, lid from a 50 mL conical tube (rim removed), forceps, and scissors. The lung tissue is carefully resected into smaller blocks with fine forceps and sterile scissors (Figure 1A,B). Next, to embed the lung tissue blocks, the bottom of the cup is filled with a small amount of the agarose, a lid is placed on the surface of the agarose, and the cup is stored at 4 &#176;C for 5 min (Figure 1C). Approximately 1 mL of liquid agarose is added to the lid, and the lung tissue block is placed onto the lid. The agarose is left for 2-3 min to semi-solidify at room temperature (Figure 1D). Once a lung block is held in place by the semi-solidified agarose, liquid agarose is then poured into the cup until the lung tissue block is fully submerged. This is stored at 4 &#176;C for 15 min (Figure 1E,F). The low-melting-point agarose embedding provides the necessary support and stiffness to the lung tissue, which helps maintain its original architecture during the slicing process. A sharp blade is used to remove excess agarose surrounding the lung tissue. A uniform layer of approximately 5 mm thickness is left around the tissue (Figure 1G). The excised lung tissue block is then carefully glued onto the tissue holder (Figure 1H). The vibratome buffer tray is filled with PBS, ice is placed in the surrounding ice bath, and the specimen disk installed in the buffer tray (Figure 1I). The vibratome cutting parameters set on the panel were: slice thickness: 200 &#181;m; frequency: 100 Hz; amplitude of the blade: 1.3mm; forward speed of the blade: 0.02-0.03 mm/s, which depends on tissue stiffness (Figure 1J). Finally, staining is carried out on free-floating slices (Figure 1K-M). 
Immunofluorescence staining was carried out in 200 &#181;m sections from each species for smooth muscle actin (SMA, a smooth muscle cell and myofibroblast marker), elastin (extracellular matrix protein) and Lycopersicon Esculentum lectin (LEA), which binds to bronchoalveolar epithelial cells. The lung is a structurally heterogeneous tissue and as such, differential SMA and elastin distribution is observed across structures, including alveolar ducts, blood vessels, intrapulmonary bronchioles, and alveoli from mice, pigs, and humans (Figure 2A-L). A high-resolution rendering of a 20 &#181;m volume of a bronchiole shows how fine structures within the lung can be examined at a semi-three-dimensional level (Supplemental Video S1). As a quantitative example, utilizing image tracing, we show how the size of the alveoli differs between samples (n = 20 alveoli) (Figure 2L,M).
To show how it is possible to quantitatively utilize these advanced imaging data, we compared type 2 pneumocyte (TII) cell distributions in human lung tissue from an adult and an infant. The human-specific type 2 alveolar cell antibody, HTII-280, has a strong affinity to the surface of human type 2 cells (Figure 3A-F). These cells can further be viewed in the three-dimensional space of a single alveolus using volumetric imaging (Supplemental Video S2). To quantify the TII number between the adult and infant samples, we processed the HTII-280 count across 10 random fields of view (fov). Between these samples, the infant sample yielded a significantly higher TII cell count (Figure 3G). However, the infant sample also exhibited a significantly higher scaffold coverage than the adult, which could explain the higher cell number (Figure 3H). To account for this, we then assessed the number of TIIs based on scaffold coverage, as cells per mm2 of tissue, which still maintained a significantly higher number of TIIs in the infant sample (Figure 3I). Thus, the higher TII count in the infant sample is not due to increased scaffold coverage and highlights the importance of assessing cell distributions in the lung based on the area of the scaffold, not the overall fov.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65943/65943fig01.jpg" /> 
Figure 1: Protocol for cutting lung tissue slices. (A) The solutions and materials: a 3% (w/v) agarose solution obtained by dissolving 1.5 g of low-melting-point agarose in 50 mL of sterile phosphate-buffered saline; a plastic cup; a lid from 50 mL conical tube; forceps, and scissors. (B) The lung tissues were dissected by using fine forceps and sterile scissors. (C) Fill the bottom of the cup with a small amount of agarose and place the lid on the surface of the agarose; then, store it at 4 &#176;C for 5 min. (D) Add 1-2 mL of liquid agarose to the lid slowly, place the lung tissue chunk onto the lid carefully, and store it at 4 &#176;C for 2 min. (E,F) Pour the liquid agarose into the cup until the lung tissue chunk is fully submerged, store it at 4 &#176;C for 15 min, and then break the plastic cup gently. (G) Use a sharp blade to remove excess agarose surrounding the lung tissue, leaving a uniform layer of approximately 5 mm thickness around the tissue. (H) The excised lung tissue block is then carefully glued onto the tissue holder. (I) Fill the vibratome buffer tray with PBS, place ice in the surrounding ice bath, and install the specimen disk into the buffer tray. (J) Set cutting parameters on the panel of the vibratome; slice thickness: 200 &#956;m, frequency: 100 Hz, amplitude of the knife: 1.3 mm, and forward speed of the blade of 0.02-0.04 mm/s. (K-M) Representative images of a free-floating slice still embedded in agarose and ready for immunofluorescence staining. <a href="https://www.jove.com/files/ftp_upload/65943/65943fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65943/65943fig02.jpg" /> 
Figure 2: Immunofluorescence images of lung structure in human, pig, and mouse. (A-L) Representative images of an alveolar duct, blood vessel, bronchiole, and alveoli in lung tissue slices from mouse, pig, and human, respectively. SMA (shown in green) is a marker for smooth muscle cells and is observed in the vascular and bronchial walls of lung tissue. Elastin (shown in red) forms part of the extracellular matrix of the lungs. (M) Quantification of alveolar size by selecting 20 random positions on each lung tissue slice for both human, pig, and mouse samples. Histograms were utilized to illustrate the distribution of alveolar size within each group. Scale bars = 50 &#181;m (A-F, H-L), 100 &#181;m (G). Kruskall-Wallis Test. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. Abbreviations: DAPI = 4&#8242;,6-diamidino-2-phenylindole; LEA = Lycopersicon Esculentum lectin; SMA = smooth muscle actin; ms = mouse; hmn = human. <a href="https://www.jove.com/files/ftp_upload/65943/65943fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65943/65943fig03.jpg" /> 
Figure 3. Type 2 pneumocyte distribution in human adult and infant samples. (A,B) Representative images of adult and infant human lung tissue stained for HTII-280 (shown in red), CD31 (shown in green), and LEA (shown in gray). (C,D) Representative images of adult and infant human showing type 2 pneumocyte distribution alone. (E,F) Single-cell resolution representative images of an individual type 2 pneumocyte from adult and infant human lung samples. (G) Quantification of the number of type 2 pneumocytes between adult and infant samples. (H) Quantification of lung tissue scaffold area coverage (% vs air) in adult and infant samples. (I) Quantification of type 2 pneumocyte per mm2 of tissue in adult and infant samples. Scale bars = 5 &#181;m (E,F), 100 &#181;m (A-D). n = 10 fov per sample. Unpaired t-test or Mann-Whitney Test. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. Abbreviations: DAPI = 4&#39;, 6-diamidino-2-phenylindole; LEA = Lycopersicon Esculentum lectin; HTII = human type II cells; CD31 = Platelet endothelial cell adhesion molecule-1/cluster of differentiation 31; fov = field of view. <a href="https://www.jove.com/files/ftp_upload/65943/65943fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video S1: Video of 20 &#181;m imaging volume of a single porcine airway stained using DAPI, LEA, SMA, and elastin showing a high-resolution view of the structure of airway wall elements. Abbreviations: DAPI = 4&#8242;,6-diamidino-2-phenylindole; LEA = Lycopersicon Esculentum lectin; SMA = smooth muscle actin. <a href="https://www.jove.com/files/ftp_upload/65943/Sup_vid1.mp4" target="_blank">Please click here to download this File.</a>
Supplemental Video S2: Video of 30 &#181;m imaging volume of a single alveolus stained using DAPI, LEA, and HTII-280 showing type 2 pneumocyte distribution across the structure.&#160;Abbreviations: DAPI = 4&#8242;,6-diamidino-2-phenylindole; LEA = Lycopersicon Esculentum lectin; SMA = smooth muscle actin. <a href="https://www.jove.com/files/ftp_upload/65943/Sup_vid2.mp4" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Studying Spatial Protein Expression Using Agarose Embedded Lung Tissue Sections at JoVE.com

Author Spotlight: Studying Spatial Protein Expression Using Agarose Embedded Lung Tissue Sections 

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.

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 ...

automated-vibratome-sectioning-agarose-embedded-lung-tissue-for

Research

JoVE Journal

Medicine

4.8K Views.  Lund University. 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. 

Video: Author Spotlight: Studying Spatial Protein Expression Using Agarose Embedded Lung Tissue Sections 

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Cancer Research

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Immunology and Infection

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Biology

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Preserving Fungal Spore Localization in Lungs for Fluorescent Microscopy Analysis

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3&#916; arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.
Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and ...

Assessing Pulmonary Microvasculature with Isolectin B4 Staining in Mice

Quantifying Pulmonary Microvascular Density in Mice Across Lobules

The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood flow regulation, and gas exchange. In murine models, lung lobes exhibit significant differences in size, shape, location, and vascularization, yet existing methods lack consideration for these variations when quantifying microvascular density. This limitation hinders the comprehensive study of lung microvascular dysfunction and the potential remodeling of microvasculature circulation across different lobules. Our protocol addresses this gap by employing two sectioning methods to quantify pulmonary microvascular density changes, leveraging the size, shape, and distribution of airway branches across distinct lobes in mice. We then utilize Isolectin B4 (IB4) staining to label lung microvascular endothelial cells on different slices, followed by unbiased microvascular density analysis using the freely available software ImageJ. The results presented here highlight varying degrees of microvascular density changes across lung lobules with aging, comparing young and old mice. This protocol offers a straightforward and cost-effective approach for unbiased quantification of pulmonary microvascular density, facilitating research on both physiological and pathological aspects of lung microvasculature.

Author Spotlight: Advances in Quantifying Microvascular Density in Aging Murine Lungs

Here, we describe a simple and economical protocol to perform unbiased quantification of pulmonary microvascular density for whole mice lung tissue using single staining of Isolectin B4.

The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood ...

Whole Lung Agarose Inflation: A Method of Preparing  Lungs for Ex vivo Live Imaging

Source: Van den Bijgaart R.J.E. et al., <a href="https://www.jove.com/v/53741/ex-vivo-live-imaging-of-lung-metastasis-and-their-microenvironment">Ex vivo&#160;Live Imaging of Lung Metastasis and Their Microenvironment</a>, J. Vis. Exp. (2016)
This video describes a method of preparing lungs for ex vivo live imaging to study interactions between cancer cells and stromal cells, using agarose for inflation. In the sample protocol, agarose inflation procedure is described before observing live cellular interactions.

Source: Van den Bijgaart R.J.E. et al., Ex vivo&#160;Live Imaging of Lung Metastasis and Their Microenvironment, J. Vis. Exp. (2016)
This video describes ...