Name: Functional Assessment of Mouse Gingival Epithelial Barrier Using Fluorescent Tracers (Video) | JoVE Journal
Uploaded: 2026-02-27T13:35:00.000+00:00
Duration: 7 min
Description: Learn a topical application method using fluorescent dextrans to assess mouse gingival epithelial barrier integrity. This approach enables quantification of tracer penetration for studying oral inflammation and disease models.

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This study was supported by the National Natural Science Foundation of China (grants 32030010, 31972908, 81991500, 81991502).

All experimental procedures were approved by the Ethics Committee of Animal Research, Peking University Health Science Center, and complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). During the process, the experimental animals were carefully treated to minimize their pain and discomfort.
The details of each work step for in vivo permeability assessment in mouse gingival epithelia are described below (Figure 1A). Here, we provided an example of detecting epithelial barrier function in both ligation- and Porphyromonas gingivalis (P. gingivalis)-induced mouse periodontitis models. All personnel received comprehensive training in laboratory animal welfare and aseptic technique.
1. Animal handling
<ol>
	<li>Throughout the study, house animals under standard specific pathogen-free (SPF) conditions with a 12 h light/dark cycle and provide food and water ad libitum.</li>
	<li>Preparation and administration of anesthetics and tracers
	<ol>
		<li>Maintain sterile conditions throughout the protocol and use tools cleaned with 75% ethanol.</li>
		<li>Use male wild-type (WT) C57BL/6J mice in the mature adult age group of 8-10 weeks for the experiment. Divide the mice into three groups: a control group (untreated) and two experimental groups (ligation- and P. gingivalis-induced mouse periodontitis models). 
		NOTE: In grouping, mice with similar body weight and week age should be selected to reduce the influence of age and weight on gingival epithelial permeability. Ligation-induced and P. gingivalis-induced mouse periodontitis models represent commonly used mouse models of periodontitis. For detailed modeling protocols, refer to the literature on ligation-induced15 and P. gingivalis-induced16 mouse periodontitis models.</li>
		<li>Select fluorescent tracers with non-overlapping spectra, such as FITC-labeled dextran (40 kDa; FD40) and rhodamine B-labeled dextran (70 kDa; RD70), to ensure distinct signal detection using corresponding FITC and rhodamine filters.</li>
		<li>Dilute the tracers. To follow this protocol, mix FD40 (1 g/L) and RD70 (1 g/L) in a 2% carboxymethylcellulose gel and store at 4 &#176;C.</li>
		<li>Intraperitoneally inject 1.25% tribromoethanol (200 mg/kg body weight, 15-17 &#181;L/g body weight) to anesthetize the mice or anesthetize according to the recommended guidelines by the&#160;local institution. 
		NOTE: Tribromoethanol solutions are light-sensitive. Tert-amyl alcohol is a flammable, volatile, and irritating organic solvent. All procedures involving preparation, handling, and administration must be performed in a chemical fume hood or an extremely well-ventilated area, strictly away from any ignition sources. Personnel must wear appropriate personal protective equipment (PPE), including gloves, chemical splash goggles, and a lab coat. Waste disposal: Waste tribromoethanol solution, tert-amyl alcohol, and all contaminated supplies (e.g., syringes, pipette tips, gloves) must be collected and disposed of as hazardous chemical waste in strict accordance with your institution&#39;s Environmental Health and Safety (EHS) regulations and protocols for flammable and toxic chemicals.</li>
	</ol>
	</li>
	<li>During anesthesia, maintain the core body temperature at 37 &#177; 0.5 &#176;C using a feedback-controlled heating pad. Apply eye lubricant to prevent corneal drying.</li>
	<li>Hold the oral cavity open with a mouth gag and apply a &#160;5 &#956;L to 50 &#956;L mixture of two or more paracellular permeability tracers with different molecular weights topically to the labial gingival surface of the molar region (Figure 1B). Leave for 30 min. If the animal swallows the mixture, reapply it.</li>
	<li>Isolation of teeth and periodontal tissues
	<ol>
		<li>After 30 min of application of permeability tracers, euthanize mice by cervical dislocation. Fix the mice on cardboard, placing them in a supine position, and tape their limbs.</li>
		<li>Following fixation, open the oral cavity to isolate the maxillary teeth and periodontal tissues. Gently clear away the surrounding tissues and rinse the specimen thoroughly with PBS.</li>
		<li>Place the teeth and periodontal tissues on a glass slide, with modeling clay positioned on both sides to facilitate secure mounting of a coverslip over the sample.</li>
	</ol>
	</li>
</ol>
2. Single-photon microscope setup
<ol>
	<li>Carefully position the dissected mouse teeth and periodontal tissues onto the custom-designed sample holder, ensuring stable placement for imaging.</li>
	<li>Acquire images using an upright single-photon microscope equipped with a 25&#215;/NA 1.0 water-immersion objective, focusing on the mounted support region beneath the teeth and periodontal tissues. 
	NOTE: Fluorescence signals should be visible immediately after removing the tracer gel.</li>
</ol>
3. Maxillary teeth and periodontal tissues imaging
<ol>
	<li>Begin imaging promptly after selecting the region of interest on the buccal surface of the second molar.</li>
	<li>Acquire Z-stack images with a step size of 1 &#181;m, starting from the maxilla gingival surface to a depth of 100-200 &#181;m, for three-dimensional (3D) reconstruction.</li>
	<li>Perform Z-series imaging to evaluate epithelial barrier permeability in the gingiva. Configure the software parameters in the referenced software as follows:
	<ol>
		<li>In the File menu, create a new folder and assign it a descriptive name for the dataset.</li>
		<li>Under the Acquisition tab, set XY format to 512 &#215; 512 with a resolution of 0.99 &#181;m/pixel.</li>
		<li>Set the Zoom factor to&#160;1.</li>
		<li>Set excitation wavelengths to 488 nm (FD40) and 561 nm (RD70), with emission windows of 500-550 nm and 570-620 nm, respectively. 
		NOTE: Gain values were set to 50 (488 nm) and 60 (561 nm), respectively.</li>
		<li>For Z-series and 3D imaging, select ND Acquisition mode. Set Z-step to 1 &#181;m (adjust as needed).</li>
	</ol>
	</li>
	<li>Once parameters are configured, select Live mode to visualize the gingival area in both individual and merged fluorescence channels within the imaging window.</li>
	<li>Click Run Now to start acquisition after parameter configuration.</li>
</ol>
4. Permeability measurement
<ol>
	<li>Divide the fluorescence intensity of FD40 and RD70 into different channels. No background subtraction is required; merge the Z-axis slices. Then, use ImageJ software to calculate the fluorescence intensity to evaluate the permeability and indicate the epithelial barrier function.
	<ol>
		<li>Click File and select Open to open the maxilla picture taken under the single-photon microscope.</li>
		<li>Go to the Image menu and change the image type to 8-bit format for analysis. 
		NOTE: Images captured by fluorescence microscopes and confocal systems are generally in RGB format and cannot be used directly for fluorescence intensity measurement. They need to be split into individual channels or monochrome images should be directly imported.</li>
		<li>Open the Analyze menu, choose Set Measurements, and check Area, Mean, and IntDen. Confirm by clicking OK.</li>
		<li>Select the channel to be analyzed and adjust the threshold: Go to Image | Adjust | Threshold. The system will automatically apply a default value and algorithm, typically using Red to indicate selected regions. 
		NOTE: As fluorescent images typically feature black backgrounds, also select the Dark Background option.</li>
		<li>Set measurement parameters: Go to Analyze and select Set Measurements. In the dialog box, check Mean gray value and Limit to threshold. Finally, click OK. 
		NOTE: It is recommended to check Display label so the image name appears in the results table, making it easier to identify the experimental group.</li>
		<li>Perform measurement: Select the Freehand Selections tool. Use the cursor to manually and precisely trace the outline of the fluorescent signal region on the gingival tissue side. Click Analyze | Measure. The results table will appear, where IntDen represents the fluorescence intensity.</li>
	</ol>
	</li>
	<li>Copy the results to a spreadsheet for calculations and import for graphing.</li>
	<li>Calculation of Relative Fluorescence Intensity: First, calculate the mean fluorescence intensity of the control group. Then, divide the fluorescence intensity value of each experimental group by the control group mean to obtain the relative fluorescence intensity.</li>
</ol>

<ol><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+on+the+protection+of+gingival+epithelial+barrier+by+interleukin-22+through+regulating+microbiota+and+E-cadherin+expression%5BTitle%5D)+AND+%22Zhonghua+Kou+Qiang+Yi+Xue+Za+Zhi%22%5BJournal%5D)">Study on the protection of gingival epithelial barrier by interleukin-22 through regulating microbiota and E-cadherin expression.</a> Zhonghua Kou Qiang Yi Xue Za Zhi. 59 (7), 653-662 (2024).</li>
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<li>Costello, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+community+variation+in+human+body+habitats+across+space+and+time%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Bacterial community variation in human body habitats across space and time.</a> Science. 326 (5960), 1694-1697 (2009).</li>
<li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+normal+bacterial+flora+of+the+oral+cavity%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Defining the normal bacterial flora of the oral cavity.</a> J Clin Microbiol. 43 (11), 5721-5732 (2005).</li>
<li>Meyle, J., Chapple, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+aspects+of+the+pathogenesis+of+periodontitis%5BTitle%5D)+AND+%22Periodontol+2000%22%5BJournal%5D)">Molecular aspects of the pathogenesis of periodontitis.</a> Periodontol 2000. 69 (1), 7-17 (2015).</li>
<li>Oriuchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porphyromonas+gingivalis+lipopolysaccharide+damages+mucosal+barrier+to+promote+gastritis-associated+carcinogenesis%5BTitle%5D)+AND+%22Dig+Dis+Sci%22%5BJournal%5D)">Porphyromonas gingivalis lipopolysaccharide damages mucosal barrier to promote gastritis-associated carcinogenesis.</a> Dig Dis Sci. 69 (1), 95-111 (2024).</li>
<li>Takeuchi, H., Nakamura, E., Yamaga, S., Amano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porphyromonas+gingivalis+infection+induces+lipopolysaccharide+and+peptidoglycan+penetration+through+gingival+epithelium%5BTitle%5D)+AND+%22Front+Oral+Health%22%5BJournal%5D)">Porphyromonas gingivalis infection induces lipopolysaccharide and peptidoglycan penetration through gingival epithelium.</a> Front Oral Health. 3, 845002(2022).</li>
<li>Abe, T., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+ligature-induced+periodontitis+model+in+mice%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Optimization of the ligature-induced periodontitis model in mice.</a> J Immunol Methods. 394 (1-2), 49-54 (2013).</li>
<li>Chatterjee, A., Bhattacharya, H., Kandwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probiotics+in+periodontal+health+and+disease%5BTitle%5D)+AND+%22J+Indian+Soc+Periodontol%22%5BJournal%5D)">Probiotics in periodontal health and disease.</a> J Indian Soc Periodontol. 15 (1), 23-28 (2011).</li>
<li>Shin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photon-efficient+imaging+with+a+single-photon+camera%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Photon-efficient imaging with a single-photon camera.</a> Nat Commun. 7, 12046(2016).</li>
<li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+endothelial+tight+junctions+to+predict+and+protect+thoracic+aortic+aneurysm+and+dissection%5BTitle%5D)+AND+%22Eur+Heart+J%22%5BJournal%5D)">Targeting endothelial tight junctions to predict and protect thoracic aortic aneurysm and dissection.</a> Eur Heart J. 44 (14), 1248-1261 (2023).</li>
<li>Cai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foxf1+maintains+endothelial+barrier+function+and+prevents+edema+after+lung+injury%5BTitle%5D)+AND+%22Sci+Signal%22%5BJournal%5D)">Foxf1 maintains endothelial barrier function and prevents edema after lung injury.</a> Sci Signal. 9 (424), ra40(2016).</li>
<li>Hou, A., Mohamed Ali, S., Png, E., Hunziker, W., Tong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transglutaminase-2+is+critical+for+corneal+epithelial+barrier+function+via+positive+regulation+of+claudin-1%5BTitle%5D)+AND+%22Ocul+Surf%22%5BJournal%5D)">Transglutaminase-2 is critical for corneal epithelial barrier function via positive regulation of claudin-1.</a> Ocul Surf. 28, 155-164 (2023).</li>
<li>Kitsuki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+junctional+epithelial+permeability+in+trpv4-deficient+mice%5BTitle%5D)+AND+%22J+Periodontal+Res%22%5BJournal%5D)">Enhanced junctional epithelial permeability in trpv4-deficient mice.</a> J Periodontal Res. 55 (1), 51-60 (2020).</li>
<li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porphyromonas+gingivalis+impairs+oral+epithelial+barrier+through+targeting+grhl2%5BTitle%5D)+AND+%22J+Dent+Res%22%5BJournal%5D)">Porphyromonas gingivalis impairs oral epithelial barrier through targeting grhl2.</a> J Dent Res. 98 (10), 1150-1158 (2019).</li>
<li>Lemaitre, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Periodontal+tissue+regeneration+using+syngeneic+adipose-derived+stromal+cells+in+a+mouse+model%5BTitle%5D)+AND+%22Stem+Cells+Transl+Med%22%5BJournal%5D)">Periodontal tissue regeneration using syngeneic adipose-derived stromal cells in a mouse model.</a> Stem Cells Transl Med. 6 (2), 656-665 (2017).</li>
<li>Takahashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gingival+epithelial+barrier%3A+regulation+by+beneficial+and+harmful+microbes%5BTitle%5D)+AND+%22Tissue+Barriers%22%5BJournal%5D)">Gingival epithelial barrier: regulation by beneficial and harmful microbes.</a> Tissue Barriers. 7 (3), e1651158(2019).</li>
<li>Ye, P., Chapple, C. C., Kumar, R. K., Hunter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+patterns+of+E-cadherin%2C+involucrin%2C+and+connexin+gap+junction+proteins+in+the+lining+epithelia+of+inflamed+gingiva%5BTitle%5D)+AND+%22J+Pathol%22%5BJournal%5D)">Expression patterns of E-cadherin, involucrin, and connexin gap junction proteins in the lining epithelia of inflamed gingiva.</a> J Pathol. 192 (1), 58-66 (2000).</li>
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The authors have no conflicts of interest.

The gingival epithelium serves as an essential physical barrier against bacterial invasion, while oral microorganisms exhibit dual regulatory capacity through either disrupting barrier integrity with virulence factors or promoting its maintenance. These biological interactions ultimately determine tissue homeostasis or disease progression17,18. Building on this foundation, we developed an innovative methodological approach to further investigate these mechanisms....

The gingival epithelial barrier constitutes a critical defensive interface in the oral cavity, preserving tissue integrity by selectively regulating substance transport while preventing microbial invasion1. Composed of stratified squamous epithelium interconnected by tight junction complexes, this dynamic structure precisely controls paracellular permeability to restrict the non-selective penetration of microorganisms, macromolecules, and solutes2. Multiple factors can compromise the integrity of the gingival barrier. The oral cavity hosts the second most diverse microbial community in the human body, with over 700 bacterial species colonizing tooth surfaces, gingival crevices, and oral mucosa3,4. The epithelial cells lining the gingival tissues are persistently exposed to this dense microbial environment5.
Virulence factors secreted by periodontal pathogens (e.g., Porphyromonas gingivalis), including lipopolysaccharides, have been shown to disrupt epithelial barrier function, thereby triggering inflammatory responses in gingival tissues and driving pathological progression6,7. Inflammatory mediators and mechanical stressors further exacerbate barrier dysfunction, increasing susceptibility to oral diseases8. Oral probiotics may help prevent periodontal disease by promoting the stability of the gingival epithelial barrier. Nevertheless, the biological interactions between microorganisms and gingival epithelial cells in modulating barrier function remain largely unelucidated9. Thus, the development of reliable methods to assess epithelial barrier function is of paramount importance.
Single-photon laser-scanning microscopy, recognized for its high resolution and established detection system, has proven particularly valuable for examining ex vivo tissue samples10. Advanced imaging methodologies significantly improve the evaluation of gingival barrier properties. The superficial nature of gingival tissues, with epithelial cells located approximately 100 &#181;m from the surface, makes it ideally suited for such imaging approaches. Our research has established an ex vivo assessment technique utilizing fluorescence-labeled dextrans of varying molecular weights as paracellular permeability tracers. Combined with single-photon laser-scanning microscopy, this method enables accurate quantification of gingival epithelial barrier function, providing insights into the mechanisms of periodontal disease and potential therapeutic interventions.
The protocol established in this study, combining localized gel-based tracer delivery with three-dimensional ex vivo imaging, is specifically optimized to address the unique challenges of assessing the gingival epithelial barrier. First, existing intravenous tracer injection approaches (Table 1) are unable to precisely assess the permeability of localized minute epithelial sites to external agents11. Second, traditional histological approaches require decalcification and sectioning, which disrupt tissue architecture and are labor-intensive and time-consuming12,13,14. In contrast, our protocol utilizes a highly adhesive gel to achieve effective tracer retention on the complex local surface. Furthermore, through a 3D image, it ensures precise, non-destructive quantification of tracer penetration depth and distribution within the gingival epithelium, thereby fully preserving the spatial architecture of the tissue. Consequently, this method is particularly suitable for high-resolution spatiotemporal assessment of gingival epithelial barrier function within the complex and confined oral environment.

<table><tbody><tr><td>40 kDa FITC-labeled dextran</td><td>Sigma Aldrich</td><td>78331</td><td></td></tr><tr><td>70 kDa rhodamine B-labeled dextran</td><td>Sigma Aldrich</td><td>R9379</td><td></td></tr><tr><td>Blunt tissue separation nickel</td><td>Bejinghuabo Company</td><td>NZW28</td><td></td></tr><tr><td>carboxymethylcellulose</td><td>MREDA</td><td>M049898</td><td></td></tr><tr><td>Disposable sterile syringe</td><td>Zhiyu Company</td><td></td><td>1 mL</td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td></td><td></td></tr><tr><td>Microtubes</td><td>Axygen</td><td>MCT-150-C</td><td>1.5 mL</td></tr><tr><td>NIS-Elements AR Image Software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Phosphate buffered saline 1x</td><td>Servicebio</td><td>G4207-500</td><td></td></tr><tr><td>Single photon microscope Nikon A1 MP+</td><td>Nikon</td><td></td><td></td></tr><tr><td>Tissue scissors</td><td>Bejinghuabo Company</td><td>M286-05</td><td></td></tr></tbody></table>

functional assessment of mouse gingival epithelial barrier using fluorescent tracers

Functional Assessment of Mouse Gingival Epithelial Barrier Using Fluorescent Tracers

The gingival epithelial barrier serves as a critical frontline defense in oral immunity and homeostasis. An intact epithelial barrier effectively prevents the penetration of harmful substances from the oral environment, whereas its disruption is closely associated with the pathogenesis of conditions such as periodontitis and oral lichen planus. Here, we established a topical application-based method to assess the integrity of the gingival epithelial barrier in mice. First, a mixture of fluorescently labeled dextrans with different molecular weights (40 or 70 kDa) in a 2% sodium carboxymethylcellulose was applied topically to the labial gingival surface of the molar region. After a 30 min incubation period, the maxilla was dissected and fixed in a customized holder for immediate imaging using a single-photon microscopy system. This approach enables visualization and quantification of tracers of different sizes across the epithelial layers, providing a functional readout of gingival epithelial barrier integrity applicable to studies of oral inflammation, wound healing, and disease modeling.

According to the standardized protocol, mount the maxillary teeth and periodontal tissues (including gingiva, periodontal ligament, alveolar bone, and cementum) of a mouse onto a custom-made holder (Figure 1C). Microscopic examination revealed green (FD40) and red (FD70) fluorescence signals in the gingiva, demonstrating tracer permeation across the epithelial barrier. After identifying the region of interest under the ocular lens, images were acquired using the software. In the control grou...

The goal of this protocol is to provide a standardized approach for quantitatively assessing the integrity of the murine gingival epithelial barrier by topically applying fluorescence-labeled dextrans of varying molecular sizes on the gingival surface in vivo, followed by ex vivo imaging using a single-photon microscopy system.

The gingival epithelial barrier serves as a critical frontline defense in oral immunity and homeostasis. An intact epithelial barrier effectively prevents ...

This is a standardized protocol for quantitatively assessing gingival epithelial barrier integrity. This protocol is useful for studying barrier dysfunction diseases and testing barrier restorative drugs in gingiva. To begin, divide male wild type C57BL/6J mice into three groups consisting of an untreated control group, a ligation-induced periodontitis group, and a Porphyromonas gingivalis induced periodontitis group.Select fluorescent tracers with non-overlapping spectra, such as FITC-labeled dextran and rhodamine B labeled dextran. To dilute the tracers, mix one gram per liter each of FD40 and RD70 in 2%carboxymethylcellulose gel. Store the prepared mixture on ice.After confirming the anesthetic depth by toe pinch reflex, use a mouth gag to hold the oral cavity of the anesthetized mouse open. Apply 50 microliters of the prepared tracer mixture topically to the labial gingival surface of the molar region. Leave the mixture in place for 30 minutes and reapply if swallowed.After 30 minutes of tracer application, fix the mouse in a supine position on cardboard and secure the limbs with tape to immobilize the animal. Next, open the oral cavity of the euthanized mouse and isolate the maxilla. Gently clear away surrounding tissues and rinse the specimen thoroughly with PBS.Place the isolated maxilla on a glass slide. Then place modeling clay on both sides to stabilize and secure the cover slip over the sample. Carefully position the dissected maxilla onto the custom designed sample holder to ensure stable placement for imaging.Using an upright single photon microscope equipped with a 25X water immersion objective. Focus on the mounted support region beneath the maxilla and acquire images. Select the region of interest on the buccal surface of the second molar within the maxilla and begin imaging promptly.Acquire Z-stack images with a step size of one micrometer, starting from the gingival surface to a depth of 100 to 200 micrometers for three-dimensional reconstruction. In the software, click file and select new folder to create a new folder for the data set and assign a descriptive name. Under the acquisition tab, set the XY format to 512 by 512 with a resolution of 0.99 micrometers per pixel.Then set the zoom factor to one. Next, set the excitation wavelengths to 488 nanometers and 561 nanometers. Configure emission windows to 500 to 550 nanometers and 570 to 620 nanometers respectively.For Z series and three-dimensional imaging, select ND acquisition mode. Set the Z step to one micrometer and adjust if necessary. After configuring parameters, select live mode to visualize the gingival area in individual and merged fluorescence channels.Then click run now to start image acquisition. To measure permeability, separate the fluorescence intensity of FD40 and RD70 into different channels. Merge the Z-axis slices without background subtraction.Then use ImageJ software to quantify fluorescence intensity to assess epithelial barrier permeability. Click file and select open to open the maxilla image in ImageJ. Then press image and change the image type to 8-bit format for analysis.Click analyze, select set measurements. Then check area, mean, and integrated density, and click okay to confirm. Then select the desired channel and adjust the threshold by clicking image, choosing adjust, and selecting threshold.Enable the dark background option to account for black backgrounds. Click analyze and select set measurements. Then check mean gray value and limit to threshold, and confirm by clicking okay.Using the freehand selections tool, manually trace the outline of the fluorescent signal region on the gingival tissue side. Click analyze and select measure to obtain fluorescence intensity values where integrated density represents the fluorescence intensity. Copy the measurement results into a spreadsheet for calculations and graphing.Calculate the mean fluorescence intensity of the control group in the spreadsheet. Divide each experimental group's fluorescence intensity value by the control group mean to obtain the relative fluorescence intensity. In the control group, both fluorescent tracers were largely confined to the gingival surface.In both ligation and Porphyromonas gingivalis treated groups, significant extravasation of both FD40 and RD70 into the gingival epithelium was observed. Semi-quantitative analysis demonstrated significantly increased fluorescence intensity of both FD40 and RD70 in the ligation and Porphyromonas gingivalis treated groups. 3D imaging revealed more extensive and diffuse tracer fluorescence distribution around the gingival vasculature in the ligation and Porphyromonas gingivalis treated groups compared to controls.This protocol quantifies mouse gingival paracellular barrier permeability and integrity. This protocol can be combined with micro-CT for multimodal image analysis. Further studies can explore barrier repair drugs and molecular mechanisms in oral diseases.

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Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

 Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Learn a topical application method using fluorescent dextrans to assess mouse gingival epithelial barrier integrity. This approach enables quantification of tracer penetration for studying oral inflammation and disease models.

Functional Assessment of Mouse Gingival Epithelial Barrier Using Fluorescent Tracers

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