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

Summary

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

Abstract

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 ≥5 points. Hematoxylin and eosin (H&E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of RORγt and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Introduction

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'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 drugs^3,4,5,6.

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 signals^7,8. In comparison to conventional IF, mIF offers several advantages: (1) it can detect weakly expressed antigens that are challenging to identify with conventional IF^9,10; (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.

Protocol

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 ± 2 °C) and relative humidity (55% ± 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

1. 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.
2. 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'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.
3. Nasal challenge: Administer 50 µL of 5% OVA via nasal drops using a 100 µL pipette (Figure 1B) on the day following the basal sensitization, repeating this procedure for 7 days (Figure 1C).
   ​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.

2. Behavioral scoring of rats

1. 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).
   ​NOTE: The confirmation of successful AR modeling is based on achieving a total score of ≥5 points¹⁴.

3. Acquisition of nasal mucosa samples

1. 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).
2. 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).
3. 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.

4. Pre-processing of nasal mucosa samples

1. 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.
   1. 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).
2. 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.
   1. 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.
3. Embedding: place the melted wax block into the embedding box (see Table of Materials) and store it in a -20 °C freezer. Once the wax solidifies, remove it and trim the wax block.
4. Slicing: insert the trimmed wax block into a paraffin microtome (see Table of Materials) and cut it into slices with a thickness of 3 µm. Place the slices in a 40 °C water bath on a slide spreader. Flatten the tissue, then lift it with a glass slide and bake it in a 60 °C oven.
   1. After the water has evaporated, and the wax is properly set, remove the slides and store them at a temperature of 25 °C.

5. H&E staining of nasal mucosal tissue

1. 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.
2. Hematoxylin staining: dispense 100 µL of hematoxylin staining solution (see Table of Materials) for 3-5 min using a 100 µL pipette, rinse for 5-10 s, add 100 µL of 0.5% hydrochloric acid ethanol for 10-30 s, rinse for 5-10 s, add 100 µL 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.
3. Eosin staining: use a 100 µL pipette to dispense 100 µL of eosin staining solution (see Table of Materials) for 5 min and rinse for 30 s.
4. 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).
5. Placing the stained section on the microscope: position the stained section on the microscope, adjust the microscope's focus until the image is clearly visible, set the magnification to 40x, and capture the image.

6. Multicolor immunostaining of nasal mucosal tissue

1. Dewaxing and hydration: follow the same as mentioned in step 5.1.
2. 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.
   1. 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.
3. 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).
   1. 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.
4. Blocking: apply 5% goat serum within the circle and incubate for 30 min.
5. 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 °C overnight.
   NOTE: Add a small amount of water to the humid chamber to prevent antibody evaporation.
6. 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&L, see Table of Materials) within the circle. Incubate at room temperature for 50 min in the dark.
7. Preparation of Tyramide dilution solution: combine 100 µL of 3% H₂O₂ with 100 mL of 1x TBST (see Table of Materials).
8. Addition of TSA working solution: Mix 1 mL of Tyramide diluent with 2 µL of CY3-Tyramide to prepare TSA working solution. Apply 100 µL 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 °C in the dark; it is valid for up to 24 h.
9. Repeat Steps 6.2-6.8: switch to a 1:200 dilution of RORγt (FITC fluorescent dye) and then to a 1:200 dilution of TICAM1 (TYP620 dye) (see Table of Materials).
10. 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.
11. 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.
12. 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).
13. Microscopy: place the stained section on the microscope, adjust the microscope'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).

Results

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 ≥5 points (Table 2).

H&E staining results on the 21^st day of modeling revealed that in the control rats, the nasal mucosa's e...

Discussion

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's population¹ and carries substantial economic costs, causing annual losses of up to 30-50 billion euros in EU countries¹⁸. Moreover, se...

Materials

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