The authors express their gratitude for the invaluable support received from the public experimental platform at West China School of Pharmacy. Special appreciation is extended to Wendong Wang for providing critical and highly valuable advice on pathology. This research has been made possible through the funding from the Science and Technology Department of Sichuan Province (grants 2023NSFSC0130 and 2023NSFSC1992) and &#34;the Fundamental Research Funds for the Central Universities&#34; to TJ.

All the experiments were carried out following the ethical guidelines of the Sichuan University Animal Research Committee (No K2023023).
1. Preparation of paraffin sections for mouse lung lobes
<ol>
	<li>Acquire lung tissue from the mouse.
	<ol>
		<li>Choose male C57Bl/6 mice at 6 weeks and 16 months to assess the microvascular density in mouse lungs across different age groups.</li>
		<li>Administer 100 mg/kg of pentobarbital sodium via intraperitoneal injection in mice. Euthanize the mice by cervical dislocation after anesthesia. Open the chest cavity and obtain lung tissue.</li>
	</ol>
	</li>
	<li>Prepare paraffin sections.
	<ol>
		<li>Fix the tissue by placing it in a 4% paraformaldehyde solution that is 10 times the volume of the tissue to preserve its morphology and structure and prevent degradation. 
		NOTE: A recent new method was developed to inflate air during vascular perfusion-fixation in murine lungs. This method is reported to preserve better morphology and location of the airway, alveolar cells, and interstitium, making them more suitable for lung histologic studies. This method is recommended for laboratories with the corresponding equipment and consumables with in-house-made instruments8.</li>
		<li>Cut the lung lobe to determine the embedding direction (Figure 1).
		<ol>
			<li>After 3 h of fixation, separate the five pulmonary lobes (under direct observation of the eyes) and embed the cranial, middle, and accessory lobes of the right lung separately, with sections oriented in the direction of the vertical lobe-to-bronchus connection.</li>
			<li>Cut the caudal lobe of the right lung into 2 pieces at 4 mm from left to right in the direction perpendicular to the lung lobe-bronchus connection, and embed the slices in a wax block with the cut surface facing upward.</li>
			<li>Make three cuts in the left lung lobe. Make the first cut transversely above the main bronchial junction, 2 mm below the top. Make the second cut transversely above the main bronchial junction, 5 mm below the top. Make the third cut at the end of the lobe, 8 mm below the top. Discard the uppermost piece of tissue and encase the remaining three pieces in a wax block with the cut surface facing upward. 
			NOTE: When embedding multiple lung pieces together, a microscope wiping paper can be used to wrap them before placing them in a tissue embedding box to continue the dehydration process.</li>
			<li>Re-fix the tissue in the fixative for 24 h.</li>
		</ol>
		</li>
		<li>Place the tissue block in a container and rinse it under flowing water for 15 min.</li>
		<li>Sequentially place the tissue in the following solutions: 65% ethanol for 30 min, 75% ethanol for 30 min, 85% ethanol for 30 min, 95% ethanol I for 60 min, 95% ethanol II for 60 min, absolute ethanol I for 60 min, absolute ethanol II for 60 min, 70% ethanol for 120 min, 80% ethanol for 120 min, 90% ethanol for 120 min, 95% ethanol for 120 min, absolute ethanol I for 120 min, and absolute ethanol II for 120 min.</li>
		<li>Place the tissue in xylene I for 30 min and xylene II for 30 min.</li>
		<li>Place the tissue in soft wax below 54 &#176;C for 1 h, hard wax I at 58 &#176;C for 1 h, and hard wax II at 58 &#176;C for 30 min.</li>
		<li>Select a suitable mold size, fill it with an appropriate amount of liquid paraffin, and use forceps to position the tissue in the embedding frame in the center of the mold in the correct direction. Place the mold and tissue in the freezing area of the embedding machine and gently press the tissue with forceps.</li>
		<li>When the paraffin starts to whiten slightly, quickly position the uncovered embedding frame on top of the mold and perform the second wax injection, sealing the holes at the bottom of the embedding frame. Transfer the entire mold to the freezing table for molding.</li>
		<li>After securing the wax block onto the slicer and revealing the smooth and flat cut surface through rough and fine trimming, rotate the microtome&#39;s wheel crank to cut slices with a thickness of 4 &#181;m.</li>
		<li>Smoothly place the removed sections onto the water surface in sequential order. Once the sections are fully extended, separate the broken pieces at the junctions between each section.</li>
		<li>Tilt a glass slide underneath the section and, upon contact, slowly and evenly lift the slide out of the water surface in a vertical motion. The sections will adhere to the glass slide.</li>
	</ol>
	</li>
</ol>
2. Immunofluorescence staining for detection of pulmonary microvasculature
<ol>
	<li>Place the paraffin-embedded slices in a 65 &#176;C oven for 60 min.</li>
	<li>Place the slices on a slide rack and carry out the following steps in staining jars: immerse xylene 1, xylene 2, and xylene 3 for 15 min each, followed by immersion in xylene/ethanol (1:1) for 5 min, 100% ethanol #1, 100% ethanol #2, 85% ethanol, 75% ethanol, and distilled water for 5 min each.</li>
	<li>Dilute the modified sodium citrate antigen retrieval solution 50-fold with deionized water. Preheat the microwave on high power until boiling, then place the slide in the antigen extract. Allow the slide to boil in the antigen extract for 15 min and then cool naturally to room temperature.</li>
	<li>Wash the slides twice with PBS for 5 min each. Permeabilize with 0.25% Triton twice for 10 min each.</li>
	<li>Encircle the tissue with an immunohistochemistry pen and incubate that with 5% BSA at room temperature (RT) for 1 h. Wash the slides once with PBS for 5 min.</li>
	<li>Perform Alexa-647 Fluor conjugated IB4 and DAPI staining.
	<ol>
		<li>Keep the IB4 on ice and dilute it 1:50 with 1% BSA. Remove excess water around the circle, add 50-100 &#181;L of the diluted IB4 to each tissue section, and incubate overnight at 4 &#176;C (from this step onwards, perform all procedures in the dark).</li>
		<li>Wash the slides three times with PBS for 5 min each. Permeabilize with 0.25% Triton for 10 min.</li>
	</ol>
	</li>
	<li>Dilute DAPI (10 &#181;g/mL) with PBS at a ratio of 1:10 and add 50-100 &#181;L to each tissue section at RT for 5 min.</li>
	<li>Wash the slides three times with PBS for 5 min each.</li>
	<li>Apply a drop of anti-fade mounting medium to the slides, avoiding light exposure. Air dry the slide, then observe and capture images of the nucleus and target signals under a fluorescence microscope.</li>
</ol>
3. Quantification of pulmonary microvascular density
<ol>
	<li>Acquire images.
	<ol>
		<li>Observe lung microvascular density signals and DAPI signals under a 10x objective for each lung lobe in both the young and old age groups.</li>
		<li>Standardize the light source intensity and exposure time for each channel based on the observation results. Capture the dual-channel images covering the entire area of each lung lobe. Save the image in ND2 format with two channels (Figure 2).</li>
	</ol>
	</li>
	<li>Quantify using Image J (Supplementary Figure 1).
	<ol>
		<li>Launch ImageJ software.</li>
		<li>Import indicated images to ImageJ, then proceed to load and select them. Split the channels accordingly. Load a file in ND2 format for both DAPI and IB4 channels.</li>
		<li>Navigate to Analyze &#62; Set Scale &#62; Configure the Image Parameters as 0.48&#160;&#181;m/pixel (depending on the photography parameters) &#62; Global &#62; OK.</li>
		<li>Set the same display range to adjust the image presentation effect. Select Image&#62; Adjust &#62; Brightness/Contrast &#62; Set. Set display range for each of the two channels separately, IB4: 0-10000; DAPI: 0-20000.</li>
		<li>To eliminate the influence of background signals, proceed with the following steps: Click Process &#62; Math &#62; Subtract; Subtract the background signal based on the signal values detected by the Magic Wand tool on the background, IB4: 3000; DAPI:300.</li>
		<li>To select the threshold value that best aligns with the original image, go to Image &#62; Duplicate.</li>
		<li>Select both original and duplicate Image &#62; Type &#62; 8 bit.</li>
		<li>Select an appropriate threshold value to precisely identify the lung microvasculature. Click on Image &#62; Adjust &#62; Threshold &#62; Intermodes&#160;(choose the most realistic threshold against the original diagram), tick &#62; Dark Background &#62; Apply.</li>
		<li>Eliminate IB4 positive signals from large blood vessels in the mouse lung, including arteries and veins: Use the Magic Wand tool to select larger blood vessels compared to the duplicated image, then click Delete&#160;(repeat that until the IB4 positive signals in the endothelial cells of larger blood vessels are removed). 
		NOTE: IB4 staining signals are detected in endothelial cells of arteries and veins in the mouse lung. To achieve a specific examination of pulmonary microvasculature occurrences, it is recommended to uniformly remove the signal foci in endothelial cells of larger blood vessels.</li>
		<li>Count the number of target signals, click Analyze &#62; Set Measurements, and tick Area, Limit to Threshold, and Display Label. Then select Analyze Particles and set Size: 0-infinity; Circularity: 0.00-1.00; Show: Overly Masks; tick Summarize and click OK.</li>
		<li>Choose Summarize Results &#62; File &#62; Save as.</li>
	</ol>
	</li>
	<li>Data analysis and statistics
	<ol>
		<li>Match and organize the number of IB4 positive foci and DAPI positive foci, respectively, at the same image into a unified table.</li>
		<li>Summarize the total number of IB4 positive foci and DAPI positive foci for each lobe in indicated young or age groups.
		<ol>
			<li>Calculate the percentage of IB4 positive foci (%) by dividing the total number of IB4 positive foci by the total number of DAPI staining foci in each lobe by different groups. Present the number of IB4 positive foci, the number of DAPI staining foci, and the percentage of IB4 positive foci (%) as shown in Figure 3A.</li>
		</ol>
		</li>
		<li>Launch the data analysis software and create a new column table. 
		NOTE: GraphPad Prism 9 was used here for statistical analysis.</li>
		<li>Sequentially name them from Group A to Group J as &#34;Young cranial lobe&#34;, &#34;Old cranial lobe&#34;, &#34;Young middle lobe&#34;, &#34;Old middle lobe&#34;, &#34;Young caudal lobe&#34;, &#34;Old caudal lobe&#34;, &#34;Young accessory lobe&#34;, &#34;Old accessory lobe&#34;, &#34;Young left lobe&#34;, &#34;Old left lobe&#34;. Then, input the corresponding IB4 positive foci (%) into the table (Supplementary Table 1).</li>
		<li>Utilize unpaired t-tests for pairwise comparison to assess significant differences in the corresponding regions between the two age groups. Conduct a total of five comparisons for five lobes.</li>
		<li>Create a graph for result visualization (Figure 3B). Opt for a bar chart and include statistical analysis in the figure.</li>
	</ol>
	</li>
</ol>

Assessing Pulmonary Microvasculature with Isolectin B4 Staining in Mice

<ol>
	<li>Jia, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf-2+promotes+angiogenesis+through+a+srsf1/srsf3/srpk1-dependent+axis+that+controls+vegfr1+splicing+in+endothelial+cells.">Fgf-2 promotes angiogenesis through a srsf1/srsf3/srpk1-dependent axis that controls vegfr1 splicing in endothelial cells.</a> BMC Biol. 19 (1), 173 (2021).</li><li>Schneider, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+lung:+Physiology,+disease,+and+immunity.">The aging lung: Physiology, disease, and immunity.</a> Cell. 184 (8), 1990-2019 (2021).</li><li>Rafii, S., Butler, J. M., Ding, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiocrine+functions+of+organ-specific+endothelial+cells.">Angiocrine functions of organ-specific endothelial cells.</a> Nature. 529 (7586), 316-325 (2016).</li><li>Lipskaia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+telomerase+in+p21-positive+cells+counteracts+capillaries+rarefaction+in+aging+mice+lung.">Induction of telomerase in p21-positive cells counteracts capillaries rarefaction in aging mice lung.</a> bioRxiv. , (2022).</li><li>Hoshino, M., Takahashi, M., Aoike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+vascular+endothelial+growth+factor,+basic+fibroblast+growth+factor,+and+angiogenin+immunoreactivity+in+asthmatic+airways+and+its+relationship+to+angiogenesis.">Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis.</a> J Allergy Clin Immunol. 107 (2), 295-301 (2001).</li><li>Ackermann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vascular+endothelialitis,+thrombosis,+and+angiogenesis+in+covid-19.">Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19.</a> New Engl J Med. 383 (2), 120-128 (2020).</li><li>Wertheim, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+pulmonary+microvascular+endothelial+cells+ex+vivo:+Implications+for+pulmonary+arterial+hypertension,+and+a+caution+on+the+use+of+commercial+biomaterials.">Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials.</a> PLoS One. 14 (2), e0211909 (2019).</li><li>Thomas, S. M., Bednarek, J., Janssen, W. J., Hume, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Air-inflation+of+murine+lungs+with+vascular+perfusion-fixation.">Air-inflation of murine lungs with vascular perfusion-fixation.</a> J Vis Exp. (168), e62215 (2021).</li><li>Ramasamy, S. K., Kusumbe, A. P., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tissue+morphogenesis+by+endothelial+cell-derived+signals.">Regulation of tissue morphogenesis by endothelial cell-derived signals.</a> Trends Cell Biol. 25 (3), 148-157 (2015).</li><li>Amersfoort, J., Eelen, G., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulation+by+endothelial+cells+-+partnering+up+with+the+immune+system.">Immunomodulation by endothelial cells - partnering up with the immune system.</a> Nat Rev Immunol. 22 (9), 576-588 (2022).</li><li>Niethamer, T. 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The authors declare that they have no competing interests.

The study of pulmonary microvascular density holds significant implications for understanding pulmonary physiological processes and also for defining biomarker(s) for respiratory diseases. The pulmonary circulation boasts an extensive capillary surface area enveloped by a slender layer of endothelial cells. The harmonious juxtaposition of these cells and alveolar epithelial cells gives rise to a fragile alveolar-capillary membrane specifically designed to facilitate the intricate process of gas exchange...

Endothelial cells (ECs) are a special type of cell located on the inner lining of blood vessels, covering the entire arterial and venous tree and playing a crucial role in maintaining the stability of blood vessels and organs1. The lungs are highly vascularized organs and play essential physiological and pathological roles in the lungs, such as forming the vascular wall, regulating blood flow, facilitating gas exchange, modulating inflammatory responses, controlling platelet activity, secreting regulatory substances involved in vascular growth, repair, and maintaining coagulation balance.
Lung microvascular endothelial cells (LMECs) are specific endothelial cells of pulmonary tissue, particularly in the microvasculature (capillaries) of the lungs, distinguishing them from the more generalized arterial and venous endothelial cells in the lungs. These cells have various functions, including regulating vascular tone, controlling vascular permeability, participating in the regulation of inflammatory responses, and regulating thrombus formation. They play a crucial role in pulmonary circulation, regulating gas exchange and the transport of nutrients, and are involved in various physiological and pathological processes related to the lungs, likely for aging2. Moreover, the abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction3. Employing the conventional endothelial cell marker CD31 and spatial localization (specifically, the peripheral regions of the lungs), Larissa L.&#160;et al. observed a significant decrease in microvascular endothelial cell density in aged mice (18 months old) compared to their younger counterparts (4 months old)4. In the context of the pulmonary pathology associated with asthma, Makoto H. et al. demonstrated a substantial increase in vessel induction in bronchial biopsy specimens stained with anti-collagen IV from asthmatic patients in comparison to control subjects5. Recently, by introducing the techniques of transmission and scanning electron microscopy, Maximilian A. et al. reported a notable increase in the numeric density of features related to intussusceptive and sprouting angiogenesis in patients who died from Covid-19 or Influenza A (H1N1)6. Evidently, the abnormal microvascular genesis is linked with pulmonary dysfunction. However, there is currently no simple, economical method available for quantifying changes in microvascular density.
In murine models, the lungs are conventionally segmented into five distinct lobes: right cranial, right middle, right caudal, left cranial, and left caudal. Each lobe exhibits unique characteristics in terms of size, shape, location, and likely vascularization, contributing to efficient gas exchange and potentially synergistic regulation of pulmonary circulation. However, to the best of our knowledge, no methodologies account for the differences among these lung lobes when investigating Lung microvascular changes.
This study presents a new method for sectioning lobules in mice, utilizing IB4, a well-defined marker of lung micro-endothelial cells7, for unbiased quantitative assessment of pulmonary microvascular density. This innovative approach addresses the need for a more comprehensive understanding of microvascular alterations in murine lungs by considering the distinct properties of individual lobes of mice. As a demonstration, in aging&#160;mice, a significant reduction in pulmonary 
microvascular density is observed specifically&#160;within both the caudal lobe and left lobe.&#160;The protocol underscores the importance of integrating lobe-specific analyses into investigations of changes in the microvascular landscape of murine lungs. Notably, this method provides valuable research references for investigators seeking a comprehensive understanding of both physiological and pathological progression of lung developments and lesions, extending beyond angiogenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td>Tissue Fixative</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole</td>
			<td>MCE</td>
			<td>HY-D0814</td>
			<td>Nucleic Dyes</td>
		</tr>
		<tr>
			<td>Alexa-647 Fluor Conjugated Isolectin B4</td>
			<td>Thermo</td>
			<td>I32450</td>
			<td>Binding Microvessels</td>
		</tr>
		<tr>
			<td>Anti-fluorescent Tablet Sealer</td>
			<td>Abcam</td>
			<td>AB104135</td>
			<td>Sample Fixation</td>
		</tr>
		<tr>
			<td>Antigen Repair Fluid</td>
			<td>Biosharp</td>
			<td>BL151A</td>
			<td>Repair of Antigenic Sites</td>
		</tr>
		<tr>
			<td>Biopsy Cassette</td>
			<td>ActivFlo</td>
			<td>39LC-500-1</td>
			<td>Fixing and Positioning Tissue Samples</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>B2064-50G</td>
			<td>Sealing Solution</td>
		</tr>
		<tr>
			<td>Cold Plate</td>
			<td>Leica</td>
			<td>HistoCore Arcadia H&#160;</td>
			<td>Freezing Samples</td>
		</tr>
		<tr>
			<td>Constant Temperature Electric Drying Oven</td>
			<td>Taisite</td>
			<td>101-0AB</td>
			<td>High Temperature Repair</td>
		</tr>
		<tr>
			<td>Disposable Microtome Blade</td>
			<td>Leica</td>
			<td>14035838383</td>
			<td>Cutting Tissue Samples to Prepare Sections</td>
		</tr>
		<tr>
			<td>Embedding Molds</td>
			<td>Shitai</td>
			<td>26155166627</td>
			<td>Fixing Tissue Samples</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Kelong</td>
			<td>CAS 64-17-5</td>
			<td>Tissue Dehydration Solution</td>
		</tr>
		<tr>
			<td>Heated Paraffin Embedding Station</td>
			<td>Leica</td>
			<td>EG1150</td>
			<td>Embedding Tssue Samples in Paraffin</td>
		</tr>
		<tr>
			<td>HistoCore Water Bath</td>
			<td>Leica</td>
			<td>HI1220</td>
			<td>Flatten and Fix Tissue Samples</td>
		</tr>
		<tr>
			<td>ImageJ (Fiji)&#160;</td>
			<td>NIH</td>
			<td>1.54f</td>
			<td>Quantitative Tool</td>
		</tr>
		<tr>
			<td>Immunohistochemistry Pens</td>
			<td>Biosharp</td>
			<td>BC004</td>
			<td>Water-blocking Agent</td>
		</tr>
		<tr>
			<td>Medical Forceps</td>
			<td>Shanghai Medical Equipment</td>
			<td>N/A</td>
			<td>Grasping, Manipulating, or Moving tissue samples</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Ts2</td>
			<td>Imaging Device</td>
		</tr>
		<tr>
			<td>Mounting Media</td>
			<td>Jiangyuan</td>
			<td>Tasteless</td>
			<td>Fixing and Preserving Tissue Sections</td>
		</tr>
		<tr>
			<td>Paraffin Wax</td>
			<td>SCHLEDEN</td>
			<td>80200-0014</td>
			<td>Fixing Tissue Structure</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Beyotime</td>
			<td>C0221A</td>
			<td>Wash Buffer</td>
		</tr>
		<tr>
			<td>Pentobarbital Sodium</td>
			<td>Beijing Chemical Reagent Company</td>
			<td>Q/H82-F158-2002</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Rotary Microtome</td>
			<td>Biobase</td>
			<td>Bk-2258</td>
			<td>Preparing Slices</td>
		</tr>
		<tr>
			<td>Sterile Scissors</td>
			<td>Shanghai Medical Equipment</td>
			<td>N/A</td>
			<td>segmenting Tissue Samples</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>Shanghai Medical Equipment</td>
			<td>N/A</td>
			<td>Cutting Tissue Samples</td>
		</tr>
		<tr>
			<td>Triton&#160;</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td>Permeabilization Solution</td>
		</tr>
		<tr>
			<td>Wash-Free Slide</td>
			<td>PLATINUM PRO</td>
			<td>PRO-04</td>
			<td>Fixing Samples for Staining</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SUM</td>
			<td>XK13-011-00031</td>
			<td>Tissue De-waxing Solution</td>
		</tr>
	</tbody>
</table>

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

Quantifying Pulmonary Microvascular Density in Mice Across Lobules

Our research aims at quantifying pulmonary microvascular density in various mouse lung lobules considering anatomical differences and age-related changes using isolectin B4 staining and ImageJ analysis. Key questions that we are trying to address are how to quantify microvascular density in different lung lobules accurately, and what changes occur in microvascular density across lobules with aging. Recent advancements in pulmonary microvascular research, including improved tissue clearing and fixation techniques, advanced imaging, and software tools for accurate quantification.These developments enhance our understanding of microvascular changes in aging and related diseases, like asthma and COVID-19, offering insights for potential therapies. The main challenges are accurately locating lesions, properly sectioning lung tissues, mitigating fixative-related tissue damage, and lack of specific markers for lung endothelial cells. Traditional methods often overlook the variations inside shape and vascularization across different lung lobes.Our protocol addresses the gap by providing a comprehensive approach to sectioning lung lobes and quantifying microvascular density using isolectin before staining. The combination of IB4 staining with the free software ImageJ for advanced microvascular density analysis ensures accurate and useful results. The cellular and molecular mechanisms by which pulmonary microvascular lesions participate in the progression of lung-related diseases, and develop new strategies or targets for rescuing pulmonary microvascular function to promote lung repair and induce functional recovery.

To distinguish between the lesions in the main bronchi and small airway branches, it is crucial to ensure that the continuous structure of lesions in these two types of airways is observed. This can be achieved by following the cutting and embedding procedures outlined in Figure 1. Given the numerous lung lobes in mice, which are oriented in various directions and possess a mesh-like structure, they are more susceptible to collapse compared to other solid tissues. To achieve a clear and intu...

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 ...

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Research

JoVE Journal

Biology

Preparing Mouse Lung Tissue Sections for Isolectin B4 Staining to Evaluate Pulmonary Microvascular Density

To begin, place the mouse lung tissue in 4%paraformaldehyde, which is 10 times the volume of the tissue. After three hours of fixation, separate the five pulmonary lobes and embed the cranial, middle, and accessory lobes of the right lung separately, orienting in the direction of the vertical lobe-to-bronchus connection. Then cut the caudal lobe of the right lung into two pieces at four millimeters from left to right in the direction perpendicular to the lung-lobe-bronchus connection, and embed the slices in a wax block, with the cut surface facing upward.To make three cuts in the left lung lobe, cut transversely above the main bronchial junction, two millimeters below the top. Make the second cut transversely above the main bronchial junction, five millimeters below the top. Make the third cut at the end of the lobe, eight millimeters below the top.After discarding the uppermost piece of tissue, encase the remaining three pieces in a wax block, with the cut surface facing upward. Following refixation, rinse the tissue block under flowing water for 15 minutes. Sequentially immerse the tissue in the escalating ethanol concentrations, followed by xylene and soft wax.Fill a mold with an appropriate amount of liquid paraffin and, using forceps, position the tissue in the embedding frame at the center. Place the mold and tissue in the freezing area of the embedding machine. When the paraffin starts to whiten slightly, quickly position the uncovered embedding frame on top of the mold and perform the second wax injection, sealing the holes at the bottom of the embedding frame.Then transfer the entire mold to the freezing table. After securing the wax block onto the slicer, trim the block to reveal a smooth and flat cut surface. Rotate the microtome's wheel crank to cut slices with a thickness of four micrometers.Smoothly place the removed sections onto the water surface sequentially. Once the sections are fully extended, separate the broken pieces at the junctions between each section. Tilt a glass slide underneath the section, and, upon contact, slowly and evenly lift the slide out of the water surface in a vertical motion.

Staining the Mouse Lung Tissue Sections with Isolectin B4 and DAPI to Visualize Pulmonary Microvascular Density

To begin, obtain paraffin-embedded slice of mouse lung tissue and place it in a 65 degree Celsius oven for 60 minutes. For staining, immerse the slide with the tissue section sequentially in the staining jars containing appropriate solutions. Dilute the modified sodium citrate antigen retrieval solution 50-fold with deionized water.After preheating the antigen extract in the microwave on high power, place the slide in it for 15 minutes to boil. After the slide cools to room temperature, wash it two times with PBS for five minutes each. Then permeabilize the tissue with 0.25%triton two times for 10 minutes each.Encircle the tissue with an immunohistochemistry pen and incubate the slide with 5%BSA at room temperature for one hour. Next, dilute the isolectin B4, or IB4, with 1%BSA to a ratio of 1 to 50. After removing excess water, incubate the tissue section with 50 to 100 microliters of the diluted IB4 overnight at four degrees Celsius.Wash the slides three times with PBS for five minutes each and permeabilize with 0.25%triton for 10 minutes. Then incubate the tissue section with 50 to 100 microliters DAPI at room temperature for five minutes. After three PBS washes, apply a drop of anti-fade mounting medium to the slide, avoiding light exposure.Mount the air-dried slide under a fluorescence microscope to capture images of the nucleus and target signals. This protocol enables precise localization and observation of pulmonary microvasculature using IB4 for staining micro endothelial cells in different mouse lung lobes under a fluorescence microscope.

Unbiased Analysis of Microvascular Changes in Isolectin B4 Stained Murine Lung Lobules Using ImageJ

To begin, obtain Isolectin B4 or IB4 and DAPI-stained mouse lung tissue sections. Acquire images under a fluorescence microscope with a 10x objective. Import the images to ImageJ software and select them.Split the channels accordingly and load the file in ND2 format for DAPI and IB4 channels. Navigate to Analyze, Set Scale. Configure the image parameters, and then select Global, followed by OK.To set the same display range, go to Image, adjust brightness contrast, and click Set.Set display range for IB4 as zero to 10, 000, and that for DAPI as zero to 20, 000. To eliminate the background, click Process, followed by math. Subtract the background signal based on the values detected by the magic wand tool on the background.For selecting the best threshold value, go to Image and click Duplicate. Select both Original and Duplicate Image type to 8-bit. Next, under the Image tab, select Adjust followed by Threshold and Enter Modes.After choosing the most realistic threshold, select Dark Background and apply. Using the magic wand tool, select larger blood vessels compared to the duplicated image, then click Delete to eliminate IB4-positive signals from large arteries and veins. To count the number of target signals, click Analyze, followed by Set Measurements, and tick Area, Limit to threshold and Display label.Then select Analyze Particles and modify Size as zero to infinity, Circularity as zero to one, and Show as Overlay Masks. Tick Summarize, and click OK.Finally, choose Summary. Click File and save the results.The percentage of the IB4-positive foci in the cranial, middle, caudal, accessory, and left lung lobes could be distinguished between the young and middle-aged mice using ImageJ analysis.

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