Materials and Reagents

1.5 ml screw cap micro tube (SARSTEDT, catalog number: 72.703.600)
5 ml screw cap tube (SARSTEDT, catalog number: 60.9921.524)
27 G x ½” 1 ml Insulin syringe (Terumo, catalog number: SS-10M2713A)
26 G x ½” needle (Terumo, catalog number: NN-2613R)
23 G x 1¼” needle (Terumo, catalog number: NN-2332R)
3 ml syringe (Terumo, catalog number: SS-03L)
5 ml syringe (Terumo, catalog number: SS-05L)
80 μm syringe filter (Sartorius, Minisart^®, catalog number: 16592)
45 μm syringe filter (Sartorius, Minisart^®, catalog number: 16555)
96-well microplate (Thermo Fisher Scientific, Nunc^TM MicroWell^TM, catalog number: 260860)
Cotton wool roll (BNS Medical, catalog number: 71841-14)
Marker pen
Aluminum foil
Male Sprague Dawley rats > 10 weeks old (Animal Resource Centre, Murdoch, Australia)
Serum samples derived from ovalbumin-sensitized murine models
Evans blue (Sigma-Aldrich, catalog number: E2129-10G), store at room temperature
Ovalbumin (OVA) lyophilized powder (Sigma-Aldrich, catalog number: A5503), store at 4 °C
Sterile Water for Irrigation (Baxter, catalog number: 2F7114), store at room temperature
Sterile Water for Injection (Pfizer, catalog number: 25020010), store below 25 °C
Chloral hydrate crystalized (Sigma-Aldrich, catalog number: 23100-250G)
Sterile Phosphate buffered saline (PBS) (made in house)
Topical ophthalmic ointment (Novartis, Viscotears^®, catalog number: 1AD031591)
Pentobarbitone sodium (Virbac, Lethabarb^®, catalog number: LETH-1)
Isoflurane (Henry Schein, IsoThesia^®, catalog number: 988-3244), store below 25 °C in the dark
5.71% chloral hydrate solution (C₂H₃Cl₃O₂) (see Recipes)
1% Evans Blue dye stock solution (see Recipes)
10 mg/ml ovalbumin stock solution (see Recipes)
1:1 Ovalbumin-Evans blue dye solution for one rat (see Recipes)

Equipment

100 ml Schott bottle (Sigma-Aldrich, Duran^®, catalog number: Z305170)
500 ml Bottle Top Filter, 0.2 μm pore size (Thermo Fisher Scientific, Nalgene^® Rapid-Flow^TM, catalog number: 595-4520)
Animal hair clippers (Wahl, KM Cordless^TM, catalog number: WA-9596-212)
Warming pad (Kent Scientific Corporation, catalog number: DCT-25)
Small animal anesthesia system (Kent Scientific Corporation, SumnoSuite^®, catalog number: SS-01)
4 °C refrigerator
-20 °C freezer

Software

GraphPad Prism (GraphPad software, version 7.0a)

Procedure

Serial 1 in 2 dilutions of serum samples
1. Using a 96-well microplate, perform serial 1 in 2 dilutions of serum in PBS to a final sample volume of 55 μl.
  Notes:
  1. Serum samples are obtained from OVA-sensitized and aerosol challenged murine models.
  2. Final serum dilutions include: neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128 samples.
  3. Duplicate 55 μl PBS only samples are required as negative controls.
2. A total of 8 individual experimental samples can be analyzed per rat, in addition to duplicate negative control samples.
Male Sprague Dawley rat anesthetization
1. Place male Sprague Dawley rat in an anesthetic induction chamber and anesthetize via isoflurane for approximately 3 min.
2. Once sufficiently anesthetized, intraperitoneally inject the male Sprague Dawley rat with 4 ml 5.71% chloral hydrate solution using a 23 G x 1¼” needle and 5 ml syringe.
  Notes:
  1. This volume of 5.71% chloral hydrate solution will anesthetize an adult male Sprague Dawley rat (> 10 weeks old) for approximately 1 h.
  2. The male Sprague Dawley rat has reached sufficient deep plane anesthesia when the pedal withdrawal reflex is absent or barely noticeable.
3. Place a small amount of topical ophthalmic ointment on the eyes of the anesthetized rat to stop them drying out during the procedure.
Subcutaneous injections of serum dilutions
1. Place the anesthetized male Sprague Dawley rat on a warming pad and closely shave the entire back of the rat from the base of the neck to the hips.
2. Using a marker pen, mark an 8 x 8 dot grid covering the entire back of the rat. Space the dots approximately 1 cm apart (Figure 1).
  Notes:
  1. Do not use a blue marker as this will be hard to distinguish from the Evans blue dye.
  2. An adult male Sprague Dawley rat will fit approximately 8 individual serum samples.
  3. Include 2 additional dots below the 8 x 8 sample grid for duplicate negative control samples.
3. Starting with the highest dilution (1:128), subcutaneously inject 50 μl of the sample under the first marked dot using a 27 G x ½” 1 ml Insulin syringe. Continue down the first row of dots injecting each of the serial dilutions.
  Note: Start injections from the right-hand side of the rat if you inject with your right hand. This will avoid disturbing the injection sites as you progress across the back (Figure 1).
4. Subcutaneously inject the 7 remaining individual samples and the duplicate PBS negative control samples, as per Step C3.
  Note: Always use a new sterile syringe for each individual sample.
5. Once all samples have been injected, cover the back of the rat in cotton wool roll and place back in the housing cage.
6. Monitor the anesthetized male Sprague Dawley rat periodically until full consciousness is regained.
  
  Figure 1. Representative diagram of steps performed in Procedure C
Evans blue dye intravenous injection
1. Twenty-four hours after subcutaneously injecting the serum samples, re-anesthetize the rat as per Procedure B.
2. Place anesthetized rat on its back and intravenously inject 2 ml of the 1:1 OVA-Evans blue dye solution (see Recipes) into the penile vein using a 26 G x ½” needle and 3 ml syringe.
3. Place rat on its stomach and allow samples to incubate for 15-30 min.
4. Following injection, subcutaneous serum samples that are positive for OVA-specific IgE will develop a blue color (Figure 2). Record the highest dilution for each individual serum sample that returns a positive indication.
5. Once all data has been recorded, humanely euthanize the male Sprague Dawley rat by intravenously injecting 600 μl of Lethabarb^® Pentobarbitone sodium into the heart using a 27 G x ½” 1 ml syringe.
  
  Figure 2. Extravasation of Evans blue detection dye indicating the presence of OVA-specific IgE in the serum sample. Subcutaneously injected serial dilutions of serum samples that contain OVA-specific IgE will become blue following the intravenous injection of an OVA-Evans blue dye solution. Record the highest dilution at which a blue injection site is present for each sample (i.e., 1:4 dilution for Sample 2).

Data analysis

Plot data on a log₂ scale to account for the serial dilutions (i.e., neat = 1, 1:2 = 2, 1:4 = 3, 1:8 = 4, 1:16 = 5, 1:32 = 6, 1:64 = 7, 1:128 = 8) using a graphing program such as GraphPad prism. Depending on the number of experimental groups, statistical significance can be determined by Student’s t-test or one-way ANOVA. As detailed in the original research article studying BALB/c mice (Mincham et al., 2018; Figure 1A), by additional research conducted within our laboratory studying Brown Norway rats (Leffler et al., 2018) and in Figure 3 below comparing gender-specific responses following early life OVA sensitization and aerosol challenge (Protocol ID: 1802387), OVA-specific serum IgE titres are significantly greater in OVA sensitized and aerosol challenged animals compared to naïve control animals.

Figure 3. Enhanced serum OVA-specific IgE titers in early life OVA sensitized and aerosol challenge BALB/c mice. Comparison of male and female serum OVA-specific IgE titers as measured by passive cutaneous anaphylaxis assay. Data are presented from individual animals comparing PBS control versus OVA sensitized and aerosol challenged male (shaded) and female (white) BALB/c mice and displayed as box and whisker plots showing minimum to maximum of n ≥ 6 independent experiments. Statistical significance was determined using Student’s t-test and presented as **P < 0.01, ****P < 0.0001.

Recipes

5.71% chloral hydrate solution (C₂H₃Cl₃O₂)
1. Dissolve 5.71 g chloral hydrate in 100 ml sterile water for injection at room temperature
2. Cover (100 ml Schott bottle) with aluminum foil to protect from light and store at 4 °C
1% Evans Blue dye stock solution
1. Dissolve 1 g Evans blue powder in 100 ml PBS
2. Store at 4 °C
10 mg/ml ovalbumin stock solution
1. Dissolve 1 g OVA lyophilized powder in 100 ml sterile water for irrigation by placing lyophilized OVA powder on the water surface and allow to slowly dissolve at room temperature without stirring the solution
2. Once dissolved, filter the 10 mg/ml ovalbumin solution into a sterile 100 ml Schott bottle using a 500 ml bottle top filter
3. Aliquot the sterile 10 mg/ml OVA stock solution into 1 ml aliquots (1.5 ml screw cap micro tubes)
4. Store at -20 °C
1:1 Ovalbumin-Evans blue dye solution for one rat
1. Add 400 μl of 10 mg/ml OVA stock solution to 600 μl PBS to yield a final concentration of 4 mg/ml OVA solution in a total volume of 1 ml
2. Add 1 ml 1% Evans blue dye to 1 ml 4 mg/ml OVA solution
  Note: Filter the necessary volume of 1% Evans blue dye stock solution required for the experiment through 80 μm and 45 μm syringe filters prior to use to remove precipitation.
3. Store at 4 °C until required
  Note: Make up the 1:1 OVA-Evans blue dye solution immediately before anesthetizing the male Sprague Dawley rat.

Acknowledgments

The authors would like to acknowledge the animal technicians at the Telethon Kids Institute Bioresources facility. The original research article (Mincham et al., 2018) utilizing this protocol was funded by the National Health and Medical Research Council of Australia.

Competing interests

The authors declare no financial or non-financial competing interests related to this work.

Ethics

All animal experiments relating to this protocol were formally approved by the Telethon Kids Institute Animal Ethics Committee, which operates under guidelines developed by the National Health and Medical Research Council of Australia for the care and use of animals in scientific research.

References

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