Autoimmune hypophysitis is characterized by an enlargement of the pituitary gland and a deficiency in one or more pituitary hormones. The causative autoantigens remain unknown. Here, a protocol for preparing mouse pituitary gland proteins for use in studying this disease is demonstrated.
First pituitary glands are isolated from euthanized mice. The pituitaries are then homogenized to mechanically break the cells and release cytosolic proteins. The homogenous is clarified by low speed centrifugation and assessed for quantity by colormetric, BCA assay and quality by poly acrylamide gel electrophoresis.
Finally, the aqueous protein homogenate is mixed with an oily adjuvant. This mixture is then injected into mice to create a mouse model resembling autoimmune hypophysitis. As shown in the companion video, Four techniques have been described to induce hypophysitis in experimental animals.
The technique presented in this article that is the injection of pituitary proteins mix influenza adjuvant is the oldest one dating back to the 1960s. Other approaches have been the injection of soluble glycoproteins from the rubella virus in 1992, the stereotactic injection of an adenovirus directly into the pituitary gland in 2002, and the transplantation pituitary gland from one rat strain under the kidney capsule of another rat strain in 2010. The main advantage of the Florence's adjuvant approach is that it is less invasive nowadays, more consistent and above all, more accurate in inducing a disease that closely resembles the human counterpart.
Although being reported since the 1960s, the foreign adjuvant technique performed poorly for the induction of hypophysitis. When I started my PhD work, I could obtain only modest hypophysitis instance and severity using the published foreign adjuvant protocol. Things improve in significantly when I have an idea of increasing by tenfold the amount of pitu proteins contain in the EM emotion.
Though this methods can provide insights into the pathogenesis of autoimmune hypophysitis, it can also be used to study other autoimmune diseases for which the auto antigens remain to be identified. Generally, individuals new to this methods will struggle mainly with the last step, the preparation of the margin, because it requires manual skills, time and dedication. Visual demonstration is therefore critical to understand how a proper emulsion should be prepared.
The implication of this technique extend beyond the field of auto immunity. Knowing how to prepare the in antigen for immunization can also be used for in the production of monoclonal antibodies or for testing new vaccines. Demonstrating this technique will be S Zoo heroic Ura and Mayland al gato from my laboratory.
The mouse pituitary is a cylindrical shaped gland that sits at the base of the skull in a depression of the sphenoid bone called the terica. The gland is bordered laterally by the trigeminal nerves and anteriorly by the optic nerve chiasm. It is composed of a larger anterior lobe and smaller, intermediate, and posterior lobes.
To isolate the pituitary gland, use scissors to decapitate the euthanized mouse, then make parallel cuts from the back to the front of the skull. Lift the brain and flip it backward to expose the pituitary gland, which appears as a cylinder in the context of an isosceles triangle. Having the pituitary gland as a base, the trigeminal nerves as sides, and the optic chiasm as a vertex.
Circle the gland with the tips of closed forceps to break the surrounding meninga. Then using fine forceps, grab the pituitary from the cell of Turca and place it in a plastic tube on dry ice. For one preparation, collect 300 mouse pituitary glands.
This collection requires approximately nine person hours. Once the collection is completed, store the pituitary glands at minus 80 degrees Celsius until they're needed. Place a 15 milliliter Falcon tube in an ice bath.
Add to the tube 250 microliters of homogenization buffer. For every 100 pituitaries collected just before homogenization, add the mouse pituitary glands to the buffer. Next place the tip of the poly tron homogenizer a five millimeter diameter generator at the bottom of the Falcon tube containing the mouse pituitaries and buffer homogenize at maximum speed for 30 seconds.
Gently moving the tube up and down while keeping it on ice. Then rest the homogenate on ice for one minute. Repeat the homogenization and cooling steps two more times.
This gentle homogenization should break the cells and release the proteins without breaking the nuclei Centrifuge. The homogenate at low speed, about 1000 G for 10 minutes at four degrees Celsius to sediment, the nuclei, unbroken tissue, and insoluble materials such as connective tissue and denatured protein complexes. Taking care not to disturb the pellet.
Transfer the SNA now called post nuclear sate or PNS to a new Falcon tube store. The PNS on ice retract the pellet using the same volume of homogenization buffer as before homogenize and centrifuge as before. After pooling the two PNS samples, eloqua the solution into numerous micro centrifuge tubes.
Store the micro centrifuge tubes at minus 80 degrees Celsius until they're needed. This protein preparation is stable at minus 80 degrees Celsius, but gradually loses its potency. Thus, it is recommended to use it within six months from the preparation.
Retain one small aliquot of PNS to determine the protein yield and concentration by the biy acid assay. Then determine the protein quality by gel electrophoresis from 300 mouse pituitary glands, which average a total weight of approximately 570 milligrams. Expect a yield of 60 milligrams of pituitary proteins or one milligram of protein for every 10 milligrams of tissue.
The protein concentration is typically around 40 milligrams per milliliter. The example provided here is for immunizing 10 mice. Plan to inject each mouse with 100 microliters of emulsion containing one milligram of pituitary protein, allowing two extra mice for material losses during the preparation here, 12 milligrams of protein emulsion are prepared.
Begin by rapidly thawing the frozen protein aliquots by warming them up in your hands. Adjust the protein concentration to 20 milligrams per milliliter with PBS. Next, draw 600 microliters of the pituitary protein extract corresponding to 12 milligrams into a 2.5 milliliter Gas Tite Hamilton syringe resuspend, complete FRONS adjuvant or CFA containing five milligrams per milliliter of heat killed mycobacterium tuberculosis by manually shaking it.
Then draw 600 microliters of the CFA into another 2.5 milliliter Hamilton syringe. Attach and secure a 22 gauge micro emulsifying needle to the syringe filled with CFA. Slowly push the plunger such that the needle is filled with CFA.
Then attach and secure the other end of the needle to the other syringe filled with pituitary protein extract. Avoid trapping air bubbles inside the syringes. Slowly advance the plunger of the CFA syringe to push the oil component into the aqueous pituitary extract such that only a small amount is mixed.
Then push back the plunger of the pituitary extract syringe into the CFA syringe. Repeat this action to gradually increasing the amount of material mixed until the entire contents are mixed. This whitish and evenly distributed material represents the water and oil emulsion.
Once the emulsion is formed, repeat the full back and forth cycle at least 500 times after the final cycle, the syringes will contain a one-to-one water and oil emulsion where the pituitary protein concentration is now 10 milligrams per milliliter. The emulsion is ready to use to test the quality of the emulsion at a drop of it to a beaker of water. If the emulsion is prepared correctly, the drop will stay compact and tight on the surface.
If the emulsion is of poor quality, it will spread and split into smaller drops. The emulsion is injected the same day of its preparation into SJL mice to induce experimental autoimmune hypophysitis. This protocol is detailed in the accompanying jo of vertical.
Once the pitu gland had been collected and techniques are mastered, one can perform the protein instruction in one hour, remembering to work on fast and on ice. During this step, During the preparation of the immersion, which requires approximately one hour. Remember to work slowly and patiently in the initial mixing phase After its development.
This technique paved the way for researchers in the field of autoimmunity to explore the mechanisms through which autoreactive lymphocytes recognize and infiltrate the target organ. After watching this video, you should have a good understanding on all how to prepare a protein extract from the mouse pituitary gland and how to mix it with Crohn's aju ones to prepare an emulsion that is ready to be used as an immunogen. Don't forget that working with syringes and complete flu edge ones, which contains inactivated and dry microbacterial tuberculosis can be hazardous if you accidentally puncture yourself.
So always use G glows and dispose needles properly while performing this procedure.
