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.

<table><tbody><tr><td>Al(OH)&lt;sub&gt;3&lt;/sub&gt;</td><td>Sollerbauer Biotechnology Co., Ltd</td><td>A7130</td><td></td></tr><tr><td>75% ethanol</td><td>Anhui Yiren An Co., Ltd</td><td>20210107</td><td></td></tr><tr><td>Ammonia</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2021070101</td><td></td></tr><tr><td>Anhydrous ethanol</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2022070501</td><td></td></tr><tr><td>Anti-fluorescence quenching sealer</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>Automatic dyeing machine</td><td>Thermo scientific</td><td>Varistain Gemini ES</td><td></td></tr><tr><td>Carrier slides</td><td>Nantong Mei Wei De Experimental Equipment Co., Ltd</td><td>220518001</td><td></td></tr><tr><td>Citrate-phosphate buffer&amp;nbsp;</td><td>Servicebio biotechnology co., Ltd</td><td>G1201</td><td></td></tr><tr><td>Citric acid antigen repair solution (PH 6.0)</td><td>Xavier Biotechnology Co., Ltd</td><td>G1201</td><td></td></tr><tr><td>Coverslip</td><td>Nantong Mei Wei De Experimental Equipment Co.</td><td>220518001</td><td></td></tr><tr><td>Coverslip</td><td>Nantong Mewtech Life Science Co., Ltd</td><td>CS01-2450</td><td></td></tr><tr><td>CY3-Tyramide</td><td>Sawell Biotechnology Co., Ltd</td><td>G1223-50UL</td><td></td></tr><tr><td>DAPI</td><td>Sawell Biotechnology Co., Ltd</td><td>G1012</td><td></td></tr><tr><td>Decoloring shaker</td><td>SCILOGEX</td><td>S1010E</td><td></td></tr><tr><td>EDTA decalcification solution</td><td>Wuhan Xavier Biotechnology Co., Ltd</td><td>CR2203047</td><td></td></tr><tr><td>Electric heating blast dryer&amp;nbsp;</td><td>Shanghai Yiheng Scientific Instruments Co., Ltd</td><td>DHG-9240A</td><td></td></tr><tr><td>Embedding box marking machine</td><td>Thermo scientific</td><td>&amp;nbsp;PrintMate AS</td><td></td></tr><tr><td>Embedding machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-P5</td><td></td></tr><tr><td>Fast tissue dewatering machine</td><td>Thermo scientific</td><td>STP420 ES</td><td></td></tr><tr><td>Film sealer</td><td>Thermo scientific</td><td>Autostainer 360</td><td></td></tr><tr><td>FITC-Tyramide</td><td>Sawell Biotechnology Co., Ltd</td><td>G1222-50UL</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Sunny Optical Technology Co.Ltd</td><td>CX40</td><td></td></tr><tr><td>Foxp3</td><td>Affinity Biosciences Co., Ltd</td><td>bs-10211R</td><td></td></tr><tr><td>Freezing table</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JB-L5</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (HRP)</td><td>Liankebio Co., Ltd</td><td>GAR0072</td><td></td></tr><tr><td>Goat serum</td><td>Biosharp</td><td>BL210A</td><td></td></tr><tr><td>H&amp;amp;E staining kit</td><td>Leagene</td><td>DH0020</td><td></td></tr><tr><td>Hemostatic forceps</td><td>Shanghai Medical Devices Co., Ltd</td><td>J31010</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sichuan Xilong Science Co., Ltd</td><td>210608</td><td></td></tr><tr><td>Immunohistochemical pen</td><td>Biosharp</td><td>BC004</td><td></td></tr><tr><td>Microwave oven</td><td>Midea</td><td>M1-L213B</td><td></td></tr><tr><td>Neutral gum</td><td>Sinopharm Group Chemical Reagent Co., Ltd</td><td>10004160</td><td></td></tr><tr><td>Ovalbumin&amp;nbsp;</td><td>Sollerbauer Biotechnology Co., Ltd</td><td>A804010</td><td></td></tr><tr><td>Oven</td><td>Shanghai Yiheng Scientific Instruments Co., Ltd</td><td>DHG-9240A</td><td></td></tr><tr><td>Palm centrifuge</td><td>SCILOGEX</td><td>D1008E</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Beyotime Biotechnology Co., Ltd</td><td>P0099-100ml</td><td></td></tr><tr><td>Pathology section scanner</td><td>3DHISTECH Kft</td><td>Pannoramic SCAN&amp;nbsp;</td><td></td></tr><tr><td>PBS buffer</td><td>Biosharp</td><td>G4202</td><td></td></tr><tr><td>Pipette&amp;nbsp;</td><td>Dragon</td><td>KE0003087/KA0056573</td><td></td></tr><tr><td>Ror&amp;gamma;t</td><td>Affinity Biosciences Co., Ltd</td><td>DF3196</td><td></td></tr><tr><td>Scalpel</td><td>Quanzhou Excellence Medical Co., Ltd</td><td>20170022</td><td></td></tr><tr><td>Self-fluorescent quenching agent Sudan Black B</td><td>Bioengineering Co., Ltd</td><td>A602008-0025</td><td></td></tr><tr><td>Slicer</td><td>Thermo scientific</td><td>HM325</td><td></td></tr><tr><td>Slicing machine</td><td>Thermo scientific</td><td>HM325</td><td></td></tr><tr><td>Slide</td><td>Nantong Mewtech Life Science Co., Ltd</td><td>PC2-301</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Sichuan Academy of Traditional Chinese Medicine</td><td>SYX &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65937/65937eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; 2023-0100</td><td></td></tr><tr><td>TICAM-1</td><td>Affinity Biosciences Co., Ltd</td><td>DF6289</td><td></td></tr><tr><td>Tissue scissors</td><td>Shanghai Medical Devices Co., Ltd</td><td>J22120</td><td></td></tr><tr><td>Tissue spreading baking sheet machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JK-6</td><td></td></tr><tr><td>TYR-690 fluorescent dyes</td><td>Shanghai Rutron Biotechnology Co., Ltd</td><td>RC0086-34RM</td><td></td></tr><tr><td>Vortex mixer</td><td>SCILOGEX</td><td>SLK-O3000-S</td><td></td></tr><tr><td>Water bath-slide drier</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JK-6</td><td></td></tr><tr><td>Wax trimmer</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JXL-818</td><td></td></tr><tr><td>Xylene</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2022051901</td><td></td></tr></tbody></table>

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), ...

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Research

JoVE Journal

Medicine

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

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Immunology and Infection

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Neuroscience

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

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 ...

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

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

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Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue