Use of the Cockroach Antigen Model of Acute Asthma to Determine the Immunomodulatory Role of Early Exposure to Gastrointestinal Infection

Summary

The increased incidence of asthma over the last 50 years in developed countries has been associated with a decrease in infections acquired early in childhood. These early infections are thought to shape subsequent immune responses. Although there have been multiple clinical associations between gastrointestinal infections and decreased asthma incidence, it has been difficult to move beyond a simple correlation when studying human patients. This section describes an acute asthma model in C57BL/6 mice designed to specifically evaluate the effect of prior gastric Helicobacter colonization and inflammation in amurine model of cockroach allergen-induced asthma.

1. Introduction

In 1989, Strachan proposed the hygiene hypothesis to answer the growing epidemiological trend of the rise of industrialization, decline in infectious diseases, and subsequent increase in diseases associated with a T-helper 2 (Th2) cell response, including asthma.[1] Despite the growing evidence for this hypothesis, the immunological mechanism by which decreasing infection rates and increasing Th2 diseases occurs has not been elucidated.[2] Numerous studies have shown a role for viruses in asthma incidence. In fact, Strachan, et al. showed that newborns who were exposed to respiratory viruses had an increased incidence of asthma.[3] However, there is a growing body of research that suggests that immune system development, spurred by early colonization with bacteria, decreases the rate of asthma development later in life.[2] For example, studies have shown that children reared in a household with a dog or whose bedrooms had high endotoxin levels were at a lower risk for developing asthma.[4,5] Additionally, numerous studies have shown that living on a farm dramatically reduces the risk for asthma, presumably because of the constant levels of bacterial exposure.[6] This clearly demonstrates the benefit of exposing young children to bacteria and bacterial components in reducing asthma incidence.

In developing countries, many of the infectious diseases are known to elicit a Th1 phenotype. One of these bacteria is Helicobacter pylori, which is endemic in many developing countries. As countries become more industrialized, antibiotic therapy is more widespread and these infectious diseases decline. This is especially noted when the incidence of infection is higher in adults, since they were young when the bacterial exposure was greater, than in children who were reared under more newly established standards of infection control.[7] Interestingly, the timing of bacterial exposure is critical in whether or not the microbial acquisition helps in shaping the immune system or causing a pathogenic problem. A study done with Helicobacter pylori infection in children and adults indicated that in children less than 10 years old, IL10 and IFNγ were significantly upregulated when compared to older children and adults.[8] The IL10 increase persisted past 10 years old and was higher than that of non-infected people, indicating that this early exposure promotes a Th1 immune response and that regulation of this response persists.

The bacterial exposure component of the hygiene hypothesis has gained acknowledgment and prompted members of the American Thoracic Society to set forth the notion that bacterial manipulation is a key factor in asthma prevention. Furthermore, they charge that bacterial exposure, especially through the gastrointestinal tract, is fundamental to properly developing a Th1-skewed immune system.[2] A key component of this immune system development is the body being able to detect the presence of the bacteria. Our lab has previously shown that, in C57BL/6 mice, toll-like receptors (TLRs) 2 and 4 are significantly upregulated two weeks after birth.[9] TLRs are pathogen-associated pattern receptors. TLRs 2 and 4 are specific for Gram positive and Gram negative bacterial ligands, respectively. However, in mice born to dames on a broad spectrum antibiotic cocktail and who are then weaned onto this cocktail, this upregulation does not occur.[9] This indicates that the ability to sense bacteria is critical in immune system development. Our lab has also shown that mice who were born to parents that had been on broad spectrum antibiotics and were, themselves, maintained on this regimen, had delayed Th1/17 development.[9] These data demonstrate the need for bacterial recognition and bacterial exposure in immune system development. To evaluate the role of bacteria in the hygiene hypothesis, we have developed a model where gastritis is induced by infection with the gastrointestinal bacteria Heliobacter felis; and subsequently asthma is induced using cockroach antigen (CRA).

Helicobacter pylori infection is endemic in most developing countries, affecting at least 70% of the population and colonizing about 20% of the United States population. Physiologically, the presence of H. pylori in the gut has been shown to elicit a strong IFNγ response, which has been shown to downregulate Th2 cells.[10] Specifically, administering the neutrophil-activating protein of H. pylori, both systemically and mucosally, reduces characteristic Th2 cytokines, IgE, and eosinophilia by activating the Th1 pathway.[11] These studies show that this bacterial infection causes an overwhelming Th1 response, one that epidemiologically and physiologically have been shown to directly affect the response of the Th2 pathway. In recent years, the role of Th17 cells in Helicobacter infections has been shown to be critical in pathogenesis. Shi, et al. demonstrated that the Th17 cells are critical in acute infection, where it works with IL8 to recruit neutrophils and decrease bacterial burden.[12] Depleting these cells leads to proliferation of bacteria. Interestingly, if there is no control mechanism in place, such as regulatory T cells, IL17 is upregulated to the point where it is ineffective in pathogen clearance.[13] Data from our lab shows that in germfree mice, IL17 levels continue to climb after 8 weeks, while H. felis is never cleared. With conventionally reared C57BL/6 mice, IL17 increases initially, but decreases after 8 weeks.[14] Th1 and Th17 adaptive immunity plays a significant role in Helicobacter infections, though both must be tightly regulated.

Human studies demonstrate that polymorphisms in the TLR2 and 4 genes affect the pathogenesis of Helicobacter gastritis.[15,16] However, there have been conflicting in vitro reports about which of these TLRs are needed to recognize the bacteria. Many epidemiological studies have shown an inverse correlation with this pathogen and asthma susceptibility. Chen and Blaser demonstrated that asthma onset in children younger than 5 years old was inversely associated with seropositivity for H. pylori (OR, 0.58; 95% CI, 0.38-0.88). In the same study, seropositivity for H. pylori in children 3-19 years old was significantly inversely correlated with having a current case of asthma (OR, 0.41; 95% CI, 0.24-0.69).[17] Interestingly, with the rise of asthma, there has also been an increase in administering broad spectrum antibiotics in small children. This illustrates that, not only is there a dysregulation in global bacteria, but also that the bacteria, such as H. pylori, that were acquired during childhood that could potentially shape the immune system are being eliminated before their physiological effects can be properly achieved.[18] Additionally, H. pylori is associated with decreasing the severity of gastroesophogeal reflux disorder, which is a positive correlate of asthma.[10] Immunologically, asthma is characterized as a Th2 disease, as asthmatic patients have increased serum IgE, production of which is known to be initiated by IL-4, 5, and/or 13.[10] Interestingly, IFNγ production has been shown to hinder Th2 cytokine production; IFNγ is a downstream target of NFκB, the transcription factor induced by TLR2 and 4 activation. Thus, activating TLR2 and 4 by environmental exposure to bacterial ligands could elicit a more Th1- and Th17-skewed adaptive response and result in the downregulation of the Th2 phenotype in asthma patients.[19]

Because asthma is a complex disease, various mouse models are used to elucidate various facets of the disease. Since mice do not spontaneously get asthma, the mice must be sensitized with a specific allergen and subsequently challenged with that allergen. Many labs use BALB/c mice for their studies because these mice have a Th2-skewed immune system and a robust asthmatic response upon asthma induction. However, this model does not parallel typical human disease since most people do not have a strongly Th2 skewed immune system. Therefore, the use of C57BL/6 mice is a good model because it has a Th1 bias, which is more like the human immune system. Using C57BL/6 mice is also beneficial when studying the bacterial colonization and/or sensing in asthma, as these mice respond more strongly to bacteria than the BALB/c mice.[20] Likewise, the allergens used in asthma studies vary greatly. Historically, ovalbumin has been used to induce the asthmatic phenotype because of its ability to produce strong Th2 responses.[21,22] However, this allergen does not have clinical application, as ovalbumin is not a common allergen in humans. Therefore, more clinically relevant allergens, such as house dust mite and cockroach extracts, are becoming more frequently used.[23,24] There are two categories of asthma models: acute and chronic. The acute models are used to study the onset and beginning stages of asthma, whereas the chronic models are used to study the long term effect of asthma, such as airway remodeling. The chronic model is also helpful in studying therapies for asthma patients, since most patients already have remodeling taking place in their lungs.[24]

In this model, we chose cockroach antigen (CRA) because the cockroach is a common household pest. However, this model has only previously been evaluated in adult mice, whereas we needed a model of childhood asthma induction if the effect of early gastrointestinal infections is to be adequately tested. Therefore, we utilized 2, 4, and 6 week old mice to determine the induction of acute asthma by CRA. We concluded that, at 2 weeks, the newly weaned pups did not have a mature enough immune response to mount a Th2 acute asthma response. At 6 weeks, their immune reactions had switched to a more Th1/17 phenotype and the results were more variable. However, 4 week old C57BL/6 mice had both a robust and consistent response to CRA and can now be established as an excellent model for acute sensitization and challenge with cockroach antigen (CRA) to study the development and early stages of asthma (Figures 1 and 2).

Figure 1.

Four week old mice have more robust pathology and airway resistance than 2 or 6 week old mice. (A) Four week old mice developed more inflammation within their airways and vasculature. Although 6 week old CRA-treated mice have moderate perivascular and peribronchial inflammation, the younger 4 week old mice have much greater perivascular and peribronchial inflammation after asthma induction. The 2 week old mice have only mild perivascular and peribronchial inflammation after CRA treatment. (B-D)Representative H&E images of the 2 week old mice, 4 week old mice, and 6 week old mice, respectively. (E) Resistance (R) in the airways was consistently higher in the 4 week old mice when compared with the mock-treated, as well as (F) the treated 2 week old mice and (G) the treated 6 week old mice.

Figure 2.

Four week old mice developed higher serum asthma markers. (A) Serum IgE was consistently greater in 4 week old mice than the other two groups. (B-D) The disease in the 4 week old mice (C) appeared to be IL13 mediated, though all of the Th2 cytokines were elevated in these mice, as opposed to (B) the 2 week old mice and (D) the 6 week old mice.

2. Materials

2.1. Helicobacter Components

1. C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME)

2. Helicobacter felis ATCC 49179

3. ATCC Medium 260 Plates: trypticase soy agar, defibrinated calf blood (5% v/v)(Colorado serum Company, Denver, CO), Trimethoprim (1 mg/ml), Vancomycin (10 mg/ml), Fungizone (1% v/v). Resuspend 20 grams of Trypticase Soy Agar in 500 ml of ultrapure water (e.g. Milli-Q or equvalent) and heat with frequent agitation to boiling for one minute to completely dissolve the powder. This should be autoclaved for 15 minutes at 121°C and then cooled to 55°C in a water bath. When cooled, add 25 ml of the defibrinated calf blood using sterile technique and stir slowly to mix (see Note 1). Add 5 ml of Trimethoprim, 1.5 ml of Vancomycin, and 5 ml of Fungizone (all previously sterilized) to the mix. Immediately pour plates in a sterile hood to approximately ½ full and flame the tops to get rid of any air bubbles. Allow the medium solidify, and then store at 4°C in a sealed container for no more than one month. This makes 20-25 plates.

4. Brain Heart Infusion (BHI) Broth: Add 37 g of BHI broth to 1 L of deionized distilled water. Autoclave for 30 minutes on the liquid cycle. Let cool to 55°C in a water bath and then add the following reagents: 3 μg/ml Vancomycin (3.0 ml of a 10 mg/ml stock); 10 μg/ml Trimethoprim (10.0 ml of a 1 mg/ml stock); 1% Fungizone (10 ml); and 5% defibrinated fetal calf serum (50 ml).

5. BBL™ CampyPak™ Plus Microaerophilic System Envelopes with Palladium Catalyst (BD, San Jose, CA)

6. Histology sponges and cassettes

7. Citrosolv (Fisher)

8. Pepsin (0.25% in PBS)

9. Rabbit anti-H. felis/H. pylori antibody (Covance, Emeryville, CA)

10. Cy3 donkey anti-rabbit antibody (1:200 dilution, Jackson Immunoresearch)

11. FITC-labeled lectin N-acetyl-D-glucoaminyl-specific Griffonia simplifolica II (5 μg/mL, Invitrogen, Eugene, OR)

12. Hoechst 33258 (0.5 ug/ml; bis-Benzimide, Sigma, St. Louis, MO)

2.2. CRA Asthma Model Components

1. Cockroach antigen (Hollister-Stier, Spokane, WA) (see Note 2)

2. Incomplete Freund's Adjuvant

3. Phosphate buffered saline (PBS)

4. 2-glass syringes (5 ml each)

5. Three-way stopcock

6. 1 ml syringe with needle (32G)

7. Eppendorf tubes (1.5 ml, one per mouse)

8. Isoflurane

9. P20 & P200 pipette

10. Plastic disposable pipette dropper (one per mouse)

11. 10% buffered formalin

12. Bouin's fixative solution: This is a picric acid-formalin-acetic acid mixture that can either be made within the lab (300 ml saturated picric acid, 100 ml formaldehyde, 20 ml acetic acid) or purchased. This fixative allows better and crisper nuclear staining than 10% neutral-buffered formalin. As picric acid is extremely explosive if allowed to dry out, it is usually safer to just purchase the Bouin's fixative solution.

13. RNAlater RNA Stabilization Reagent (Ambion, Austin, TX): This is an immediate RNA stabilization and protection reagent. It allows tissue archiving without the risk of RNA degradation.

14. Methacholine: Used in the methacholine challenge, in which the subject inhales aerosolized methacholine to determine the level of bronchial hyperreactivity.

15. Ketamine: Used for the induction and maintenance of general anesthesia

16. Flexivent (Scireq, Montreal, Canada): This is a computer-controlled precision pump that controls mechanical ventilation while also obtaining measurements of respiratory mechanics.

2.3. ELISA Components

1. Immunlon 96 well plates (Thermo Fisher Scientific, Waltham, MA)

2. Goat Anti-Mouse IgE-UNLB (10 μg/ml; Southern Biotech, Birmingham, AL): For use in coating the ELISA plate.

3. Wash buffer (PBS, 0.5% Tween-20)

4. Blocking buffer (PBS, 5% bovine serum albumin (BSA))

5. Diluent buffer (PBS, 1% BSA)

6. Mouse IgE Standard (Southern Biotech)

7. Goat Anti-Mouse IgE-AP (Southern Biotech) (1:2000 in diluent buffer): For detection of IgE in serum.

8. 3N NaOH: 120g in 1000 ml of water.

9. SIGMAFAST™ p-Nitrophenyl phosphate Tablets (Sigma)

10. VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA).

2.4. RNA and QPCR Components

1. Applied Biosystems Assays-On-Demand primer/probe sets.

2. TaqMan Universal PCR Mix (PE Applied Biosystems; Foster City, CA).

3. Trizol® (Life Technologies, Grand Island, NY)

4. Turbo DNase Kit (Ambion, Austin, TX).

5. Roche Transcriptor First Strand cDNA Synthesis Kit (Roche, Pensberg, Germany)

6. Stratagene MX3000P Real-Time Cycler (Agilent Technologies)

3. Methods

3-1. Growing Helicobacter felis

1. One vial of H. felis (ATCC 49179) is inoculated onto an ATCC Medium 260 plate (see Note 3).

2. Incubate at 37°C for 2 days in an anaerobic jar with a CampyPak (activate CampyPak per instructions with water). Make sure the container has an airtight seal (see Note 4).

3. Check H. felis viability by dropping one drop of the bacterial suspension onto a microscope slide and covering with a standard coverslip. Using a 20x or 40x objective lens on a light microscope, focus on the bacteria. Make sure the majority of them are motile by watching them swim through multiple viewing fields.

4. If motile, using the broth from the plate, inoculate BHI broth (100 ml) in a flask. Secure in an anaerobic jar with a CampyPak.(see Note 5).

5. Incubate at 37°C for 18-24 h, with gentle shaking.

6. Check viability, as outlined in 3-1.3.

7. Determine the optical density of the bacteria culture (OD₄₅₀; 1 OD₄₅₀∼= 10⁹ bacteria) (see Note 6).

8. Harvest bacteria by centrifuging at 3000 g for 10 minutes, and then resuspend the pellet in glycerol: BHI freezing media (31 ml glycerol: 69 ml BHI). Store aliquots of bacteria (2×10⁹ CFU/ml) at -80°C. The frozen stock should remain viable for ∼4 months.

3-2. H. felis Innoculation

1. Day 0 and 3: Inoculate mice orally (per os (p.o.)) using a 200 ml pipette with 0.05 ml of H. felis (2 × 10⁹ CFU/ml in glycerol: BHI) using a frozen aliquot.

2. Day 7: Inoculate mice p.o. as described in 3-2.1 using freshly grown H. felis. Use the same culture technique as described above in 3-1, with the exception that a vial of frozen stock of H. felis can be used to start the culture instead of a new vial from ATCC (see Note 7).

3-3. Asthma Sensitization (Day 0)

1. Cockroach antigen (CRA) is resuspended in PBS to a final concentration of 4 mg/ml (see Note 2).

2. It is then put into one glass syringe. A second glass syringe is filled with an equal amount (v/v) of Incomplete Freund's Adjuvant (IFA).

3. Connect these 2 glass syringes to a 3-way stopcock, and then emulsify the IFA and CRA by syringe-extrusion (alternatively pushing the solution in each syringe through progressively smaller pore sizes in the stopcock) for 10-15 minutes (see Note 8).

4. The solution is finished emulsifying when a drop of the solution does not disperse on the top of ice-cold water. Use the solution immediately (see Note 9).

5. Using a non-tuberculin syringe and a 32G needle, inject the mice intraperitoneally (50 ul) and subcutaneously (50 ul).

3-4. Asthma Induction via Intranasal Challenge (Days 14, 18, 22, 26)(see Note 10)

1. Mix CRA with PBS (0.075 mg/ml) (see Note 2).

2. Anaesthetize mice individually using isoflurane (see Note 11).

3. Using a P20 pipette, drop PBS: CRA solution (10 ul, 0.75 ug) onto nostril. Once the mouse inhales the solution, drop another 10 ul onto the nostril (see Note 12).

3-5. Asthma Induction via Intratrachial Challenge (Day 28)

1. Mix CRA with PBS (0.12 ug/ml) (see Note 2).

2. Anaesthetize mice individually using isoflurane (see Note 13).

3. When the mouse begins to awaken and gasp using its diaphragm, instill PBS: CRA (50 ul, 6.0 ug) solution into the mouse's throat between gasps with a P200 pipette (see Note 14).

3-6. Sacrifice (Day 29)

1. Anaesthetize mice using isoflurane.

2. Perform cervical dislocation.

3-7. Tissue Collection

1. Mice are euthanized using 5 ml of isoflurane for approximately 30 sec and followed by cervical dislocation.

2. Blood is collected via heart puncture, allowed to clot for 20 min at room temperature (r.t.), centrifuged for 10 minutes and 1300 rpm, and the serum removed and stored at -20°C until analysis.

3. The stomach is removed and quartered. Each quarter is either fixed in Bouin's Fixative for histology, frozen at -80°C (2 quarters) for protein analysis, or stored in RNAlater for RNA analysis, as per protocol.

4. Place 1 stomach quarter flat between 2 thin histology sponges in a histology cassette. Store the cassette in Bouin's fixative for 24 hours at 4°C.Bouin's fixative is replaced with 70% ethanol every 24 hours for 48 hours (see Note 15). The tissue can be embedded in paraffin along its long, cut edge in order to get a cross section of the epithelium. One 5 μm slide is stained with hemotoxylin and eosin and scored for pathology. Other slides can be stained with various antibodies to determine helicobacter infection rates, presence of inflammatory cells, differentiated epithelial cells, proliferation, and/or apoptosis, as needed.

5. Freeze stomach quarters 2 and 3 at -80°C for protein extraction, if needed.

6. Store stomach quarter 4 in RNAlater and process as directed by the manufacture's protocol for future quantitative real-time RT-PCR (qRT-PCR).

7. The lungs are removed above the main stem branch directly below the larynx. The lungs are then separated directly below the main stem branch. Using a 25G needle and a 3 ml syringe, the left lung is perfused with 1-2 ml of formalin through the main bronchus until it is expanded but not over extended. Pressure must be maintained on the fluid in the lungs by gripping the forceps around the needle inserted into the bronchus, while gently applying pressure to the syringe. This is held for 1-2 min. Then, the lung is removed from the needle and placed into a histology cassette and stored in formalin.

8. The tissue in the cassettes is stored in formalin for 24 hours. The formalin is then replaced with 70% ethanol once every 24 hours for 2 days. The tissue can be embedded in paraffin and stained with hemotoxylin and eosin. Lung sections are then scored for pathology.

9. The right lung is stored in RNAlater as directed by the manufacture's protocol for future qRT-PCR.

3-8. Airway Hyperresponsiveness

1. Mice are anesthetized with 450 mg/kg of ketamine, and a tracheotomy tube (18G) is inserted and connected to the inspiratory and expiratory ports of a ventilator (Fexivent). Mice are ventilated at a rate of 160 breaths per minute at a tidal volume of 0.2 ml with a positive end-expiratory pressure of 2-4 cm water.

2. Increasing concentrations of methacholine (0, 10, 20, 30, 40, and 50 mg/ml) are administered via aerosolization. From 20 seconds to 3 minutes after each aerosol challenge, detailed measurements should be recorded continuously. The measurements should include resistance (R), compliance (C), and elastance (E) (see Note 16).

3-9. IgE ELISAs

1. Immunlon 96 well plates are coated with Goat Anti-Mouse IgE-UNLB (10 μg/ml) in PBS, overnight at 4°C.

2. The next day, the plate is washed with wash buffer five times. The non-specific binding sites are blocked for >1hour using blocking buffer.

3. After washing the plates five times with wash buffer, the serum samples are added. The serum from mice who received CRA is diluted 1:2 with diluent buffer, and the mock-treated mice serum samples are used neat. Mouse IgE-UNL Bis diluted to a start concentration of 2000 pg/ml, and then diluted 1:2 seven more times for the complete standard curve. The standards and samples are incubated overnight at 4°C.

4. The next day, the plate is washed 5 times with wash buffer. Goat Anti-Mouse IgE-AP (1:2000 in diluent buffer) is added and incubated at r.t. for 2 hours.

5. After washing the plate 5 times with wash buffer, SIGMAFAST™ p-Nitrophenyl phosphate Tablets are dissolved in 20 ml of deionized water and added to the plate. Plates are incubated for 30 minutes at r.t. in the dark.

6. 3N NaOH is added to stop the reaction, and the plate is read at 405 nm on a microplate reader.

3-10. RNA and RT-PCR

1. RNA Isolation: The Trizol® (phenol and guanidine isothiocyanate) method can be used to isolate the total RNA from one quarter of each stomach and the left lung.[25]

2. Before making cDNA, the RNA is processed to remove contaminating DNA using the Turbo DNase Kit. cDNA is made using the Roche Transcriptor First Strand cDNA Synthesis Kit with mRNA (2 μg) from each sample. Quantitative real-time reverse transcription polymerase chain reaction (QPCR) is performed on each sample. Primers/probes used are from Applied Biosystems Assays-On-Demand. These are used with TaqMan Universal PCR Mix. All RNA data is analyzed using the —2^ΔΔCt relative quantitation method, described in the Applied Biosystems manufacturer's protocol (see Note 17).[25-27]

3-11. Pathology Scoring

1. Stomach: One quarter stomach from each mouse that was stained using hemotoxylin and eosin should be scored on a scale of 0-3 in each of 3 categories. The scores from the 3 categories are then added together for a total score, with 0 being the lowest and 9 being the highest possible scores.[28]

   Score	Longitudinal Extent of Inflammation	Vertical Extent of Inflammation	Histological Changes
   0	none	none	none
   1	patchy	basal lamina propria only	mild loss of differentiated epithelial cells
   2	<50%	transmural	moderate loss of differentiated epithelial cells
   3	>50%	both mucosa and submucosa involved	severe loss of differentiated epithelial cells

2. Lung: Each section should be scored using a method derived from Curtis, et. al. in which the inflammation around the vasculature and the bronchial is evaluated and added together for a total inflammation score, with 0 being the lowest and 6 being the maximum.[29]

   Score	Vascular	Bronchial
   0	no inflammation	no inflammation
   1	occasional cuffing	occasional cuffing
   2	most vessels surrounded by a thin layer (1-5) of inflammatory cells	most vessels surrounded by a thin layer (1-5) of inflammatory cells
   3	most vessels surrounded by a thick layer (>5) of inflammatory cells	most vessels surrounded by a thick layer (>5) of inflammatory cells

3-12. H. felis Staining and Quantification (see Note 18).[30]

1. Deparaffinization an unstained tissue section as follows: wash the slides two times for 10 mins per wash with Citrosolv; next, wash the slides three times for 10 mins with isopropyl alcohol; and finally, rinse the slides for 5 mins with running deionized water.

2. After rehydrating the tissue in phosphate-buffered saline (PBS), pepsin (0.25% in PBS) is incubated on the slides for 10 mins at r.t..

3. After rinsing the slides in PBS, to block non-specific binding sites and to permeabilize the tissue, add PBS blocking buffer (1% bovine serum albumin, 0.3% Triton X-100) to each slide and incubated for 1 hour at r.t.

4. The slides are then washed in PBS and the tissue stained with undiluted rabbit anti-H. pylori antibody to semi-quantitate H. felis colonization. This antibody is known to cross-react with H. felis.

5. After washing the slides in PBS, Cy3 donkey anti-rabbit antibody (1:200 dilution) and FITC-labeled lectin N-acetyl-D-glucoaminyl-specific Griffonia simplifolica II (5 μg/mL) are added to the tissue and incubated for 1 hour at r.t. for detection of H. felis and mucous neck cells, respectively.

6. To counterstain the nuclei, the slides are incubated for 20 minutes at r.t. with Hoechst 33258 (0.5 ug/ml).

7. Colonization of the antrum with H. felis is evaluated on a scale of 0 to 4, where 0 = no bacteria per gland; 1 = 1 – 2 bacteria per gland; 2 = 3 – 10 bacteria per gland; 3 = 11 – 20 bacteria per gland; and 4 = >20 bacteria per gland.(33)