Measuring Local Anaphylaxis in Mice

The overall goal of this procedure is to measure changes in vascular permeability, to quantify localized allergic responses. This is accomplished by first sensitizing the animals to an allergen, such as O albumin. The second step is to challenge the ears with an intradermal injection of the allergen.

Then Evan's blue dye is injected into the tail vein. The final step is to extract the dye from the challenged ears by incubating them overnight. In form, amide a ultimately spectro photometry is used to quantify the amount of dye that entered the ear.

The main advantage of this technique over existing methods is that dye extravasation is measured by spectrometry. This means that the assay is not easily subjected to observer bias. Generally, individuals new to this method will struggle because intradermal and tail vein injections are technically challenging and require a lot of practice to achieve proficiency.

Begin with bringing an aliquot of ova stock solution to room temperature for use. When at room temperature, dilute the ova stock to a one milligram per milliliter concentration in one XPBS. Use a five milliliter polystyrene round bottom tube.

Vortex the tube at medium speed, and carefully add an equal volume of thoroughly homogenized. Inject alum to the ova solution dropwise. In parallel, prepare a negative control solution of alum mixed into PBS at a one-to-one ratio using the same technique.

Then cap the tubes and secure them to the vortex. Run the vortex at the lowest setting for half an hour with the tubes attached. When the solutions are ready, immediately proceed with sensitizing the BAB mice.

Load the ova alum mix into a one milliliter syringe. Then with a 27 gauge needle, inject 100 microliters into the peritoneum of each mouse. Repeat this for the negative control mice using the alum solution without ova over the next three weeks.

Repeat this process for a total of three injections per mouse after the last injection, wait at least two weeks before performing the local anaphylaxis assay from frozen ova stock. Start with making a five milligram per milliliter working solution in PBS. Vortex it briefly, using 5%ISO fluorine.

Anesthetize a sensitized mouse, and when it becomes immobile, dial down the iso fluorine to 3%Now load a three 10 insulin syringe with 10 microliters of the ova preparation, and cap it with a 31 gauge needle and load a second syringe with 10 microliters of one XPBS. Next wrap a 15 milliliter conical tube with transparent adhesive tape sticky side out under a dissecting microscope, secure the dorsal side of the left ear of an anesthetized mouse to the tube. Insert the needle of the PBS syringe bevel side up between the two leaves of the ear and slowly inject the full 10 microliters.

Avoid hitting blood vessels. A successful injection will result in an obvious intradermal blip. It's critical to inject the entire volume into the ear.

If any liquid leaks out of the injection site, the animal cannot be used in the analysis. Also, if the needle goes through the ear completely or scratches the surface of the ear, the assay is invalid because these trauma will confound the results Into the right ear. Use the same method to inject the ova during the three minute period.

Transfer the mouse to a restrainer and place it under a heat lamp. Then slowly inject 200 microliters of 0.5%Evans blue dye into the tail vein. Using a 27 gauge needle, rapid injection can be damaging if the vessel gets destroyed before all the dye is injected.

Continue injecting on the opposite side of the tail. Now wait 10 minutes and then euthanize the mouse with carbon dioxide, followed by cervical dislocation. Once the animal has been euthanized, use forceps and scissors to remove the ears.

Grab the ear edge with the forceps without putting pressure on the injection site. Then cut close to, but not through the cartilage ridge at the ears base. Collect as much tissue as possible.

Now taking proper precautions, aliquot out 700 microliters of form amide for each ear into a micro centrifuge tube. Transfer an ear to each tube and incubate them overnight at 63 degrees Celsius in a dry heat block, the next day most of the dye will be extracted, but the ears may appear slightly blue. Add 300 microliters of each sample in duplicate to a 96 well plate.

Avoid transferring any fur and do not form any bubbles for blanks. Load two wells with 300 microliters of form amide. Then measure the absorbance at 620 nanometers.

Subtract the values from the background and analyze the results. PBS sensitized animals did not react to the ova challenge or PBS challenge. As expected, both ears remained white.

The year receiving the PBS challenge in OVAs sensitized animals was either completely white or lightly blue, localized to the site of injection where his ears receiving the ova challenge became progressively darker for 10 minutes after the dye was administered, the IDE treated ear tissue of the PBS sensitized animals had negligible absorbance readings after both the PBS and OVA challenge. Whereas for ova sensitized animals, PBS injection generally resulted in a mean absorbance reading of 0.13. Conversely, a 50 microgram ova challenge resulted in a mean absorbance reading of 0.58.

The amount of ova used for the challenge can be adjusted within limits. For example, a 20 microgram of a challenge resulted in a mean absorbance of 0.30. However, a challenge of less than 20 micrograms did not give reliable results.

Although it is challenging once a person is proficient at the intradermal and intravenous injections, this technique can be performed on one animal in about 15 minutes With two people working together, one performing the ear injections and the other performing the tail vein injections. The procedure can be performed on 10 animals in approximately an hour. This assay is highly versatile as it can be used for both active and passive cutaneous anaphylaxis.

It can also be used for a wide variety of allergens. Don't forget that isof fluorine can have negative effects on the human reproductive system. Only use it in a well ventilated room and have a scavenging canister attached to the induction chamber to neutralize waste gas if you're pregnant.

While using isof fluorine, wear a carbon filter mask to minimize exposure.