Method intraperitoneal (ip) administration of antigen to mice

This is the most common route being technically simple and easy. The conscious mouse is manually restrained and is held in a supine position with its posterior end slightly elevated or the head can be tilted lower than the body. The needle and syringe should be kept almost parallel to its vertebral column in order to avoid accidental penetration of the viscera. The needle is pushed in at an approximately 10º angle between the needle and the abdominal surface in the lower quadrant of the abdomen, To avoid leakage from the puncture point, the needle is run through subcutaneous tissue in a cranial direction for 2–3 mm and then inserted through the abdominal wall. The recommended volume is ˂3.0 ml.

Method intramuscular (IM) administration of antigen to mice

This should usually be avoided, as mouse muscles are small. If necessary, it may be given into the thigh muscle with injection volumes ˂0.05 ml. The tip of needle should be directed away from the femur and sciatic nerve. The mouse is anesthetized or is manually restrained by another person. The needle tip is inserted through the skin and into the muscle. Aspirate briefly with the syringe before injection. If blood or body fluid reverses, stop the procedure.

Method subcutaneous (SC) administration of antigen to mice

Subcutaneous administrations are easy. As they are rarely painful a conscious mouse can usually be used. The rate of absorption is lower than from intraperitoneal or intramuscular injections. Subcutaneous administrations are made into the loose skin over the interscapular or inguinal area.

Subcutaneous administrations over the interscapular area are made as follows. The mouse is manually restrained and then placed on a clean towel or solid surface. The needle is inserted under the skin of the interscapular area tented by the thumb and forefinger and the substance then injected. A volume ˂3 ml is recommended.

Subcutaneous administration over the inguinal area is made as follows. The mouse is restrained manually and the head tilted downwards. Holding the hind leg firmly helps this procedure. The needle is inserted into the lower left or right quadrant of abdomen avoiding the abdominal midline and the substance injected. A volume of ˂0.2 ml/site is recommended. To minimize leakage, the needle should be advanced several millimeters through the subcutaneous tissue.

Method intradermal administration of antigen to mice

This route is not recommended in general and should be restricted to cases of absolute necessity. It is very difficult in the mouse due to the very thin skin. Using a fine needle is recommended. The mouse is anesthetized, the fur clipped or hair removed from an area on the back, ventral abdomen, or hind footpad, which is wiped with 70% ethanol on a gauze sponge or swab. The skin is held tautly with thumb and index finger and the needle inserted, bevel up and at a shallow angle, just under the superficial layer of epidermis. The volume should be ˂0.05 ml per site. Resistance should be felt both as the needle is advanced and as the compound is injected. A hard bleb will be seen upon successful intradermal injection of even a small quantity of fluid. If multiple sites are injected, adequate separation is necessary to prevent coalescing of lesions.

Oral (PO) In mice and rats, an antigen solution should be administered orally by means of a bulbed cannula. The cannula is pushed down into the stomach via the palatum of the mouth. Make sure that the length and width of the cannula are correct. The length can be gauged by measuring from the tip of the nose to the diaphragm, just below the sternum. This method of administration may only be deployed by experienced staff.

Intranasal (IN) This administration route using an aqueous solution is used for immunisation and inducing tolerance. An antigen in an aqueous environment is administered by means of a pipette or a micro-cannula at the entrance to a nostril or via a droplet onto the nose of an animal held in an upright position.

11. Methods of antigen administration to rabbits.

Method intravenous (IV) administration of antigen to rabbits

IV injections are administered to rabbits in the marginal ear vein. Place animal in appropriate restraint device. Shave the hair over the marginal ear vein. Clean injection site with alcohol. Insert a small bore (22 – 30 gauge) needle with a syringe or insert a similar gauge butterfly needle (needle with tubing). Gently pull back the syringe plunger to aspirate the syringe. A flash of blood in the syringe or tubing indicates proper placement. Administer substance in a steady, fluid motion. Apply pressure to the withdrawal site to stop bleeding.

Method intramuscular (IM) administration of antigen to rabbits

IM injections are administered in the thigh muscles of the hind limb. Restrain animal manually. Clean the injection site with alcohol. Insert needle into thigh muscles, and directed away from the femur avoiding the sciatic nerve. Pull back the syringe plunger to aspirate the syringe. Any blood indicates improper needle placement, and needle must be repositioned. Administer substance in a steady, fluid motion. Take care not to administer fluid too rapidly.

Method subcutaneous (SC) administration of antigen to rabbits

The rabbit should be restrained in the normal manner. With your fingers, lift the skin to make a “tent”. Disinfect the injection site and insert needle into the subcutaneous tissue. Aspirate prior to making the injection. Proper placement should yield no aspirate. Inject. Most common injection site is the loose skin around the neck and shoulder area.

12. What are the organs of the immune system are central and peripheral? What lymphoid organs contain mature T- and B-lymphocytes?

The organs of the immune system are positioned throughout the body. They are called lymphoid organs because they are home to lymphocytes, small white bloodcells that are the key players in the immune system.

There are two groups of immune system organs.

• Primary (central)--organs where immature lymphocytes develop

  • Thymus

  • Bone marrow

• Secondary (peripheral)--tissues where antigen is localized so that it can be effectively exposed to mature lymphocytes

  • Lymph nodes

  • Appendix

  • Peyer's Patches (of GI tract)

  • Tonsils

  • Adenoids

  • Spleen

  • MALT (Mucosal-Associated Lymphoid Tissue)

    • GALT (Gut-Associated Lymphoid Tissue)

    • BALT (Bronchial/Tracheal-Associated Lymphoid Tissue)

    • NALT (Nose-Associated Lymphoid Tissue)

    • VALT (Vulvovaginal-Associated Lymphoid Tissue)

13. From which bodies receive T- and B-lymphocytes? The method of isolation of B-lymphocytes from the bone marrow of mice.