This work was supported by the Sichuan Provincial Department of Science and Technology (2021YJ0175).

The experimental protocol and procedures have received approval from the Administrative and Animal Research Committee of Chengdu University of Traditional Chinese Medicine (Record number: 2022DL-010). Eight-week-old male Sprague Dawley (SD) rats, weighing 180-200 g, were commercially obtained (see Table of Materials) and housed under a natural light/dark cycle with controlled temperature (23 &#177; 2 &#176;C) and relative humidity (55% &#177; 10%). Twelve rats were randomly divided into two groups: the control group and the AR group. All rats were acclimated to these conditions and provided with free access to food and water for one week before the trial.
1. Establishment of a rat model of AR
<ol>
	<li>Preparing the sensitizing solution: suspend 30 mg of ovalbumin (OVA) and 3 g of Al(OH)3 in 100 mL of saline (see Table of Materials)14,15.</li>
	<li>Basic sensitization: grasp and hold the fully awake rat with its abdomen facing up. Disinfect the abdomen using 75% alcohol-soaked cotton balls. Use a 1.5 mL syringe to inject 1 mL of the sensitizing solution prepared in step 1.1 into the left abdominal cavity of the rats in the AR group. Repeat this injection once every two days for 14 days (Figure 1A). 
	NOTE: Ensure that the rat&#39;s head is positioned lower than its tail to prevent damage to the large and small intestines when inserting the syringe. The injection site is located 1.5 cm from the ventral midline. Rats in the control group should receive intraperitoneal injections of 1 mL of normal saline once every two days for 14 days.</li>
	<li>Nasal challenge: Administer 50 &#181;L of 5% OVA via nasal drops using a 100 &#181;L pipette (Figure 1B) on the day following the basal sensitization, repeating this procedure for 7 days (Figure 1C). 
	&#8203;NOTE: Prepare the 5% OVA solution by mixing 5 g of OVA with 100 mL of saline. For the control group, administer drops of the same volume of saline.</li>
</ol>
2. Behavioral scoring of rats
<ol>
	<li>Within 30 min after completing the final nasal challenge, observe and record the rats using a digital camera to identify symptoms such as nose scratching, sneezing, and a runny nose, based on the scoring criteria outlined in Table 1 (Figure 1D). 
	&#8203;NOTE: The confirmation of successful AR modeling is based on achieving a total score of &#8805;5 points14.</li>
</ol>
3. Acquisition of nasal mucosa samples
<ol>
	<li>Sacrificing rats: sacrifice the rats by exposing them to an overdose of CO2. Afterward, disinfect the rats with 75% ethanol (see Table of Materials). Use a scalpel to make an incision along the two corners of the mouth to expose the muscle tissue (Figure 2A). Next, cut the joints of the zygomatic bone and the mandible on both sides of the skull using tissue scissors (Figure 2B), and remove the mandible (Figure 2C).</li>
	<li>Nasal cavity exposure: separate the skin of the maxilla using a hemostat (Figure 2D) to expose the nasal cavity (Figure 2E). Cut the bony connection between the nasal cavity and the maxilla with tissue scissors (Figure 2F). Then, sever the connection between the nasal cavity and the orbital bones on both sides at the infraorbital foramen (Figure 2G) and remove the nasal cavity (Figure 2H).</li>
	<li>Fixation: place the extracted nasal cavity in 4% paraformaldehyde for fixation and store it at room temperature for 24-72 h (see Table of Materials). 
	NOTE: Ensure that the amount of paraformaldehyde fixative used is sufficient to completely cover the sections for proper fixation.</li>
</ol>
4. Pre-processing of nasal mucosa samples
<ol>
	<li>Decalcification: begin by fixing the removed tissues from step 3.1. Wash them three times with PBS for 20 min each. Follow this by washing them three times with distilled water, each time for 20 min. Subsequently, decalcify the tissues in a decalcification solution with a volume of 20-30 times that of the tissues at room temperature.
	<ol>
		<li>The decalcification solution (see Table of Materials) should maintain a pH of 7.2-7.4. Finally, place the decalcified tissue into a dehydration box. 
		NOTE: The decalcifying solution needs to be changed weekly. The decalcification process can be concluded when the tissue softens (approximately 2 months).</li>
	</ol>
	</li>
	<li>Dehydration and waxing: transfer the dehydration box to a dehydrator (see Table of Materials). Begin with 75% alcohol for 4 h, followed by 85% alcohol for 2 h, 90% alcohol for 2 h, and 95% alcohol for 1 h.
	<ol>
		<li>Subsequently, immerse the tissue in anhydrous ethanol for 30 min, repeat the process for another 30 min, use xylene for 5-10 min, repeat this step for another 5-10 min, immerse the tissue in wax for 1 h, repeat this step for another hour, and finally, immerse the tissue in wax for an additional 1 h.</li>
	</ol>
	</li>
	<li>Embedding: place the melted wax block into the embedding box (see Table of Materials) and store it in a -20 &#176;C freezer. Once the wax solidifies, remove it and trim the wax block.</li>
	<li>Slicing: insert the trimmed wax block into a paraffin microtome (see Table of Materials) and cut it into slices with a thickness of 3 &#181;m. Place the slices in a 40 &#176;C water bath on a slide spreader. Flatten the tissue, then lift it with a glass slide and bake it in a 60 &#176;C oven.
	<ol>
		<li>After the water has evaporated, and the wax is properly set, remove the slides and store them at a temperature of 25 &#176;C.</li>
	</ol>
	</li>
</ol>
5. H&#38;E staining of nasal mucosal tissue
<ol>
	<li>Dewaxing and hydration: place the slices in xylene for 20 min, repeat the process for another 20 min, absolute ethanol for 5 min, repeat this step for another 5 min, 75% alcohol for 5 min, and finally, wash for 5 min.</li>
	<li>Hematoxylin staining: dispense 100 &#181;L of hematoxylin staining solution (see Table of Materials) for 3-5 min using a 100 &#181;L pipette, rinse for 5-10 s, add 100 &#181;L of 0.5% hydrochloric acid ethanol for 10-30 s, rinse for 5-10 s, add 100 &#181;L of 0.2% ammonia water for 10-30 s, and rinse for 30 s. 
	NOTE: After hematoxylin staining, differentiation with 0.5% hydrochloric acid ethanol is necessary to remove excessive hematoxylin dye bound to the nucleus and absorbed by the cytoplasm. When eosin staining is performed, the nuclei and cytoplasm will be clearly stained. The slices will appear red or pink after differentiation with 0.5% hydrochloric acid ethanol, so rinse immediately after differentiation to remove the acid from the tissue slices to halt the differentiation. Then, use 0.2% ammonia water to enhance nuclear staining.</li>
	<li>Eosin staining: use a 100 &#181;L pipette to dispense 100 &#181;L of eosin staining solution (see Table of Materials) for 5 min and rinse for 30 s.</li>
	<li>Dehydration and sealing: immerse the slices in absolute ethanol for 5 min, repeat the process for another 5 min, repeat once more for 5 min, dimethyl for 5 min, and xylene for 5 min. Finally, seal with neutral resin (see Table of Materials).</li>
	<li>Placing the stained section on the microscope: position the stained section on the microscope, adjust the microscope&#39;s focus until the image is clearly visible, set the magnification to 40x, and capture the image.</li>
</ol>
6. Multicolor immunostaining of nasal mucosal tissue
<ol>
	<li>Dewaxing and hydration: follow the same as mentioned in step 5.1.</li>
	<li>Citrate-phosphate buffer treatment: place the citrate-phosphate buffer (pH 6.0) (see Table of Materials) into a microwave-safe container. Heat it until boiling, remove it, and then add the slides to the container. Microwave on medium heat for 8 min until boiling, turn off the heat for 8 min, and then switch to medium-low heat for 7 min.
	<ol>
		<li>After natural cooling, transfer the slices to PBS (pH 7.4), shake, and wash them three times using a decolorizing shaker, each time for 5 min. 
		NOTE: Ensure that the buffer does not evaporate during this process, and prevent the slices from drying out.</li>
	</ol>
	</li>
	<li>Blocking endogenous peroxidase: dry the slices, draw a circle around the tissue with an immunohistochemical pen (to prevent the antibody from flowing away), and apply 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9).
	<ol>
		<li>Incubate at room temperature in the dark for 15 min. After natural cooling, place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each.</li>
	</ol>
	</li>
	<li>Blocking: apply 5% goat serum within the circle and incubate for 30 min.</li>
	<li>Primary antibody addition: gently remove the blocking solution, add FOXP3 (dilution: 1:200, see Table of Materials) to the slice, and place it in a humid chamber. Incubate at 4 &#176;C overnight. 
	NOTE: Add a small amount of water to the humid chamber to prevent antibody evaporation.</li>
	<li>Secondary antibody addition: place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each. Gently dry the slices and apply the secondary antibody (Goat Anti-Rabbit IgG H&#38;L, see Table of Materials) within the circle. Incubate at room temperature for 50 min in the dark.</li>
	<li>Preparation of Tyramide dilution solution: combine 100 &#181;L of 3% H2O2 with 100 mL of 1x TBST (see Table of Materials).</li>
	<li>Addition of TSA working solution: Mix 1 mL of Tyramide diluent with 2 &#181;L of CY3-Tyramide to prepare TSA working solution. Apply 100 &#181;L of TSA working solution to each slice and incubate at room temperature in the dark for 10 min. Then, wash the slices with PBS three times, 5 min each time. 
	NOTE: Prepare and use the TSA working solution immediately, and store it at 4 &#176;C in the dark; it is valid for up to 24 h.</li>
	<li>Repeat Steps 6.2-6.8: switch to a 1:200 dilution of ROR&#947;t (FITC fluorescent dye) and then to a 1:200 dilution of TICAM1 (TYP620 dye) (see Table of Materials).</li>
	<li>DAPI counterstained nuclei: shake the slices gently to dry them, apply DAPI staining solution, and incubate at room temperature for 10 min in the dark.</li>
	<li>Quenching autofluorescence: place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each. Apply the autofluorescence quencher (see Table of Materials) to the slices, wait for 5 min, and then wash for 10 min.</li>
	<li>Blocking: place the slices in PBS (pH 7.4) and shake them on a decolorizing shaker for three cycles of 5 min each. Gently shake the slices to dry them and seal them with the anti-fluorescence quencher (see Table of Materials).</li>
	<li>Microscopy: place the stained section on the microscope, adjust the microscope&#39;s focus for clear visibility, set the magnification to 20x and 40x, and capture the image. 
	NOTE: DAPI has a UV excitation wavelength of 330-380 nm and an emission wavelength of 420 nm (blue light). FITC has an excitation wavelength of 465-495 nm and an emission wavelength of 515-555 nm (green light). CY3 has an excitation wavelength of 510-560 nm and an emission wavelength of 590 nm (red light). TYP-690 has an excitation wavelength of 630 nm and an emission wavelength of 690 nm (pink light).</li>
</ol>

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

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D., 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=Development+of+an+unsupervised+pixel-based+clustering+algorithm+for+compartmentalization+of+immunohistochemical+expression+using+automated+quantitative+analysis.">Development of an unsupervised pixel-based clustering algorithm for compartmentalization of immunohistochemical expression using automated quantitative analysis.</a> Applied Immunohistochemistry &amp; Molecular Morphology. 17 (4), 329-337 (2009).</li><li>Lu, S., 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=Comparison+of+biomarker+modalities+for+predicting+response+to+PD-1/PD-L1+checkpoint+blockade:+a+systematic+review+and+meta-analysis.">Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade: a systematic review and meta-analysis.</a> JAMA Oncology. 5 (8), 1195-1204 (2019).</li><li>Hernandez, S., 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=Multiplex+immunofluorescence+tyramide+signal+amplification+for+immune+cell+profiling+of+paraffin-embedded+tumor+tissues.">Multiplex immunofluorescence tyramide signal amplification for immune cell profiling of paraffin-embedded tumor tissues.</a> Frontiers in Molecular Biosciences. 8, 667067 (2021).</li></ol>

The authors have nothing to disclose.

Allergic Rhinitis (AR) is a non-infectious inflammatory disease of the nasal mucosa resulting from a combination of environmental and genetic factors. It has become a global health concern, impacting work efficiency, diminishing the quality of life, impairing sleep, cognitive function, and causing irritability and fatigue. AR affects 10%-20% of the world&#39;s population&#185; and carries substantial economic costs, causing annual losses of up to 30-50 billion euros in EU countries18. Moreover, se...

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa primarily mediated by specific immunoglobulin E (IgE)1,2. It is characterized by symptoms such as sneezing, a runny nose, nasal congestion, and nasal itching. With industrialization and urbanization, the prevalence of AR is gradually increasing, affecting approximately 10%-20% of the world&#39;s population1. The immunofluorescence (IF) technique is a fluorescent staining method that utilizes an antibody-antigen binding reaction. It can be employed to detect and quantify the distribution and expression levels of specific proteins in biological tissues or cells. In AR research, IF can simultaneously detect multiple targets, including AR-related cytokines, inflammatory cells, receptors, and more, facilitating the exploration of AR pathogenesis and the effects of drugs3,4,5,6.
The multicolor immunofluorescence (mIF) staining process closely resembles traditional IF, with the addition of an antibody elution step during each round of staining. This modification enables the simultaneous detection of multiple biomarkers on the same tissue section through sequential single-labeling and multiple rounds of re-staining. mIF is based on tyramide signal amplification (TSA), allowing for repeated cycles of TSA fluorescent staining and the use of microwave heating to remove antibodies while retaining fluorescent signals7,8. In comparison to conventional IF, mIF offers several advantages: (1) it can detect weakly expressed antigens that are challenging to identify with conventional IF9,10; (2) it provides high-quality staining with an improved signal-to-noise ratio; (3) it allows for the quantification of tissue-specific structures and regions of interest11; (4) multiplexing multiple pathways efficiently utilizes tissues and conserves limited pathological resources12; (5) multi-parameter analysis through mIF offers deeper insights into tissues, uncovering hidden biological information13.
Overall, mIF allows the observation of different antigen expressions and distributions within the same sample, facilitating the study of target proteins. In the future, researchers seeking to understand the expression and distribution of multiple target proteins will find this technique a valuable choice. This study demonstrates the application of mIF for staining nasal mucosa samples from rats with AR and evaluate the establishment of a rat model of AR.

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immunofluorescent labeling in nasal mucosa tissue sections of allergic rhinitis rats via multicolor immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Six SD rats were successfully induced into the AR model through OVA intraperitoneal injection and nasal challenge. AR was induced in all rats in the AR group, accounting for 100% of the group. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose. All behavioral observations scored &#8805;5 points (Table 2).
H&#38;E staining results on the 21st day of modeling revealed that in the control rats, the nasal mucosa&#39;s e...

Watch this Scientific Journal Video about Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence at JoVE.com

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

immunofluorescent-labeling-nasal-mucosa-tissue-sections-allergic

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1.5K Views.  Hospital of Chengdu University of Traditional Chinese Medicine. This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Video: Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.

High-definition Fourier Transform Infrared FT-IR Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated ...

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl4) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl4 exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl4 exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.