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

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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 

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

A Standardized Workflow for Hyperpolarized 129Xe MRI to Study Pulmonary Function

Acquiring Hyperpolarized 129Xe Magnetic Resonance Images of Lung Ventilation

Hyperpolarized 129Xe MRI comprises a unique array of structural and functional lung imaging techniques. Technique standardization across sites is increasingly important given the recent FDA approval of 129Xe as an MR contrast agent and as interest in 129Xe MRI increases among research and clinical institutions. Members of the 129Xe MRI Clinical Trials Consortium (Xe MRI CTC) have agreed upon best practices for each of the key aspects of the 129Xe MRI workflow, and these recommendations are summarized in a recent publication. This work provides practical information to develop an end-to-end workflow for collecting 129Xe MR images of lung ventilation according to the Xe MRI CTC recommendations. Preparation and administration of 129Xe for MR studies will be discussed and demonstrated, with specific topics including choice of appropriate gas volumes for entire studies and for individual MR scans, preparation&#160;and delivery of individual 129Xe doses, and best practices for monitoring subject safety and 129Xe tolerability during studies. Key MR technical considerations will also be covered, including pulse sequence types and optimized parameters, calibration of 129Xe flip angle and center frequency, and 129Xe MRI ventilation image analysis.

Author Spotlight: Standardization and Best Practices for Advancing Lung Imaging Using 129Xe MRI

Hyperpolarized 129Xe magnetic resonance imaging (MRI) is a method for studying regionally resolved aspects of pulmonary function. This work presents an end-to-end standardized workflow for hyperpolarized 129Xe MRI of lung ventilation, with specific attention to pulse sequence design, 129Xe dose preparation, scan workflow, and best practices for subject safety monitoring.