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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 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.
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 m....
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
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.......
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.......
The authors declare that they have no competing interests.
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 "the Fundamental Research Funds for the Central Universities" to TJ.
....Name | Company | Catalog Number | Comments |
4% Paraformaldehyde | Biosharp | BL539A | Tissue Fixative |
4',6-diamidino-2-phenylindole | MCE | HY-D0814 | Nucleic Dyes |
Alexa-647 Fluor Conjugated Isolectin B4 | Thermo | I32450 | Binding Microvessels |
Anti-fluorescent Tablet Sealer | Abcam | AB104135 | Sample Fixation |
Antigen Repair Fluid | Biosharp | BL151A | Repair of Antigenic Sites |
Biopsy Cassette | ActivFlo | 39LC-500-1 | Fixing and Positioning Tissue Samples |
Bovine Serum Albumin | Sigma | B2064-50G | Sealing Solution |
Cold Plate | Leica | HistoCore Arcadia H | Freezing Samples |
Constant Temperature Electric Drying Oven | Taisite | 101-0AB | High Temperature Repair |
Disposable Microtome Blade | Leica | 14035838383 | Cutting Tissue Samples to Prepare Sections |
Embedding Molds | Shitai | 26155166627 | Fixing Tissue Samples |
Ethanol | Kelong | CAS 64-17-5 | Tissue Dehydration Solution |
Heated Paraffin Embedding Station | Leica | EG1150 | Embedding Tssue Samples in Paraffin |
HistoCore Water Bath | Leica | HI1220 | Flatten and Fix Tissue Samples |
ImageJ (Fiji) | NIH | 1.54f | Quantitative Tool |
Immunohistochemistry Pens | Biosharp | BC004 | Water-blocking Agent |
Medical Forceps | Shanghai Medical Equipment | N/A | Grasping, Manipulating, or Moving tissue samples |
Microscope | Nikon | Ts2 | Imaging Device |
Mounting Media | Jiangyuan | Tasteless | Fixing and Preserving Tissue Sections |
Paraffin Wax | SCHLEDEN | 80200-0014 | Fixing Tissue Structure |
PBS | Beyotime | C0221A | Wash Buffer |
Pentobarbital Sodium | Beijing Chemical Reagent Company | Q/H82-F158-2002 | Anesthetic |
Rotary Microtome | Biobase | Bk-2258 | Preparing Slices |
Sterile Scissors | Shanghai Medical Equipment | N/A | segmenting Tissue Samples |
Surgical Scalpel | Shanghai Medical Equipment | N/A | Cutting Tissue Samples |
Triton | Solarbio | T8200 | Permeabilization Solution |
Wash-Free Slide | PLATINUM PRO | PRO-04 | Fixing Samples for Staining |
Xylene | SUM | XK13-011-00031 | Tissue De-waxing Solution |
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