• Euthanasia

• Euthanasia and mouse necropsies require prior IACUC approval. The mode of euthanasia should be chosen which minimizes pain or distress to the animal and provides biologically meaningful samples. Only personnel with appropriate AHR training may handle live animals.

  Isoflourane inhalation: This should be given in sufficient dose to cause complete loss of breathing and pulse in the mouse. Apply 1 mL to a gauze pad in a bell jar. A porous barrier should be placed over the guaze pad so the mouse does not come in direct contact with the anesthetic. (Note: Halothane inhalation in humans has been associated on rare occasions with hepatitis.  In the laboratory setting this risk is infintesimely small but its use nonetheless has been discouraged). Use gloves and an externally vented fume hood for personal protection.

  Carbon Dioxide inhalation: CO₂ is an effective agent for euthanasia and may be used if a suitable CO₂ source, and container is available. For histological purposes it has the disadvantage that it induces capillary hemorrhages in certain tissues, particularly the lungs and brain. Dry ice should not be used because it can cause frostbite in the animal and is does not provide a controlled dose of carbon dioxide.  Sufficient quantities of CO₂ should be used to induce rapid loss of consciousness and be continued to respiratory motion ceases.  CO₂ induces a metabolic acidosis and suboptimal levels may induce agitation and hyperventillation in animals.  For this reason its use has been disparaged but in practice there is little behavioral evidence of distress, when used appropriately, and it does not inferior in this respect to expensive anesthetics.

  Cervical dislocation: This should only be performed on anesthetized animals which are unresponsive to noxious stimuli, such as a foot pinch.  It is the most quick, effective and humane way to ensure the death of the animal.  It is useful to ensure that anesthetic clearance does not lead to partial recovery. (Inhaled anesthetics may decrease due to tissue redistribution or respiratory activity). Because it may introduce traumatic artifact it should not be performed in the case where CNS or pituitary hisology is needed.  Cervical dislocation of unanesthetized animals should only be performed by experienced personnel if necessary for experimental purposes and requires IACUC approval.
  

• Choice of fixatives:  Fresh tissues should be immediately placed in plastic histology cassettes and immersed in an appropriate fixative to avoid tissue autolysis. Intestine, liver, prostate and brain are particularly prone to autolysis. Other organs can be kept a short time in cold PBS. After fixation the tissues should be transferred to 70% ethanol and stored at 4°C until processed. If RNA is to be purified the tissues should be harvested quickly, placed into eppendorf tubes or plastic vials and snap frozen in liquid nitrogen.

Formalin: Formalin solution, 10% neutral buffered (Sigma). (This contains 4% w/v formaldehyde with phosphate buffers) Formalin stabilizes proteins cross links proteins and prevents decomposition. The solution may be used several until it begins to become discolored. It is good for paraffin sections and H&E stains. Tissues should be fixed overnight, or less if they are very thin (less than 2 mm).

4% paraformaldehyde: Paraformaldeyde is the anhydrous form of formaldehyde. It needs to be made fresh in PBS or stored frozen. It is slightly less harsh than formalin and is sometimes preferred for immunostaining.

Bouin's: This fixative comes prepared from Sigma and is sometimes preferred for immunostaining with certain antibodies. It's yellow color causes can cause some undesirable discoloration of tissues.

Methyl Carnoy's: (10% glacial acetic acid, 60% methanol, 30% chloroform) This is a rapid fixative which requires only a few hours to completely permeate and fix tisues. It is routinely used by pathology lab for immunohistochemistry, but like all fixatives its utility for this purpose is antigen and antibody specific. H&E stained tissue will appear very eosinophilic (reddish) in this and other alcohol based fixatives.

OCT: This clear jelly is not a fixative but is the embedding compound for frozen sections. Frozen sections work best for immunostaining but it is difficult to cut thin frozen sections and get as good of morphology as with paraffin. Tissues can be quick frozen by placing them in 1.5 mL tubes and plunging this in liquid nitrogen prior to storage at -70°C. Alternatively frozen sections can be prepared immediately by placing tissues in molds with OCT.


B5: B5 is a mercury based fixative that provides superb morphology for hematopoietic cells.  Mercury is a hazardous chemical so this fixative should be used of sparingly and must be disposed of with EHS approval in a specially designated container. 

Dissection: Record the identifying data plus the appearance and behavior of the animal. The general approach is to record the weight of the animal, each of the individual organs and the carcass after the organs are removed. If skeletal defects are suspected save the carcass and obtain a radiograph of the skeleton. Animals should be laid upon a clean paper towel and have all 4 extremities pinned to thin styrofoam or cork board.


Body Wall:  Wet the animals fur with 95% ethanol to minimize contamination with hair. Instruments should be soaked in an ethanol if cultures are to be obtained from tissues. Dedicated scissors should be used on skin and bone.  (Wash blood and tissue from instruments before soaking them in ethanol or flaming them.  Otherwise the tissue will become fixed to the instruments)

Skin: A vertical, ventral, midline incision should be cut with scissors from the neck to pubis. This should be extended laterally at both axillae and inguinal areas to form an "I" shape. Reflect the skin laterally and pin down. Abdominal skin samples should be saved as vertical thin strips (3 x 15 mm) cut with a scalpel (In this way sections will be parallel to the hair follicles)

Mammary tissue: The inguinal and axillary mammary glands are visible as yellow fat pads adherent to the underside of the skin beneath a fascial plane. They may be separatd from the skin by grasping them with forceps and sharply dissecting (i.e. with a scalpel) them away from the underlying skin with a gentle stroking motion.

Abdomen: Fresh sterile scissors should be used to open the peritoneum if cultures of MEFs are to be made. In this case pin the body wall back with the skin.

Uterus and ovary: A gravid uterus is appaarent by it's "beads on a string" appearance in the lower abdomen. The murine cervix has two horns which extend up from a cervix in the pelvis. Cut across the cervix and lift out the uterus with forceps. As the uterus is lifted fat and myometrial attachments may be separated. At the superior end of each uterine horn lies an oviduct and an ovary. The ovaries are located just below each kidney with which they share common blood vessels. Following fixation the uterus should be cut into thin cross sections with a scalpel prior to being processed into paraffin. The ovaries should be wrapped in lens paper and embedded in a block with other small organs such as the adrenal, thyroid, lymph nodes and pituitary.

Kidney: The kidney should be cut lengthwise with a scalpel through the renal pelvis prior to formalin fixation. The cut surface should be embedded face down for sectioning.

Liver: The 3 major lobes of the liver should be separated. 3-4 mm wide strips should be cut with scissors or a scalpel prior to formalin fixation.  The cut surface should be embedded face down prior to sectioning.

Adrenal: This small (2mm) pyramid shaped organ looks like a little triangle of pinkish fat on the superior pole of each kidney.  It should be wrapped in lens paper and placed in cassettes along with ovaries, pituitary, thyroid, or other small sections to avoid losing them during fixation and paraffin processing.

Spleen: The spleen should be cut lengthwise with a scalpel prior to formalin fixation. Small white specks (lymphoid follicles) can be seen.  These may be counted on the surface of the spleen (which is the basis of the CFU-S hematopoietic assay).  The cut surface should then be embedded face down prior to sectioning.

Pancreas: Pancreas is a fatty looking tissue adherent to the first part of the intestine on one end, and on the spleen on the other. It can be fixed whole.

Intestine: The intestine should be dissected clean from all mesenteric fat. The luminal contents should be gently squeezed out with the back of a pair of scissors or similar blunt object. It should be kept moist with saline and fixed quickly to avoid degeneration of the villi. Beta-gal staining is best accomplished by opening the intestine lengthwise and pinning the edges at 2 cm intervals down on a wax lined tray. After staining the intestine can be folded in pleats and placed in cassettes for formalin fixation. For longitudinal sections, cut the folded intestine lengthwise and embed with the cut surface down. Cross sections are more difficult to obtain but one method is as described for uterus.

Prostate and Testes: The testes are easily retrieved from an open abdomen by traction on the spermatic cord.  They should not be cut prior to fixation because they will rupture, instead fix them whole.  The prosate, in the mouse, contains several different lobes.  The anterior prostate is a mat of soft tissue which lies tucked into proximal fold of the large, white, convoluted, seminal vessicles.  Take care when handling the seminal vessicles or the spermatic fluid will make a mess of the dissection. They may be trimmed near their attachment to the proximal urethra.  At the base of the bladder the seminal vessicles, ductus deferens converge onto the proximal urethra.  Situated circumferentially on the proxmal urethra are a series of soft tissue protuberances.  Posteriorly, the dorsal lobe forms a pair of wing like structures which may be fused with a small protuberance more laterally, together forming the dorsolateral prostate.  On the anterior side of the urethra the ventral prostate forms one or two small bumps.  The neck of the bladder, proximal urethra and prostatic lobes may all be fixed en bloc which will facilitate proper orientation during embedding.  Alternatively the individual lobes may be separated.  If separated lobes are fixed in one cassette it is useful to mark them with dissecting ink to facilitate their identification in histological sections.

Thorax: The thorax should be opened by cutting away the rib cage with scissors. The thymus or pericardial fat may be adherent to the inner chest wall.

Thymus: The thymus has two lobes and sits on the superior and ventral aspect of the heart. It involutes in older animals and becomes increasingly surrounded by fat (older than 3 months). Under the dissecting microscope the texture of thymus can be distinguished from fat and large vessels more easily. If lacerated the contents of the thymus may spill so it should be fixed whole. Each lobe of the thymus should be embedded with the superior edge down.  If frozen sections are to be performed then molds for OCT may be formed from the back end of a VWR marker.  Insert a single lobe into the OCT superior surface down and place on a dry ice + ethanol bath.  Be very careful not to get any ethanol into the OCT or it will turn the frozen block into a slimy mess.  

Heart: The heart should be cut in cross section with a scalpel in 3 mm strips.This will create a set of "doughnuts" which should be fixed and then embedded cut surface down.Both the right and left ventricles will be visualized in this fashion.The upper heart should also be sectioned, this consists of cardiac valves, atria and the large vessels.

Lung: The lung can be cut into strips or fixed whole. Don't squeeze air out of it, that makes it harder see the normal alveoli. 

Lymph nodes:  Lymphomas will cause generalized enlargement of lymph nodes, spleen or thymus.  Be aware that infections or skin ulcers will cause enlargement of the nearby nodes and may need histology be distinguished from lymphomas. Infections or severe anemia may also cause splenomegaly.  Lymph nodes are small (1-2 mm) pale tan ovoid structures to be found in the axilla, inguinal area, and in the abdomen. They should be fixed whole. Lymphomas can be sectioned prior to fixation and embedded cut surface down. Small lymph nodes in normal areas are found in these same areas but are difficult to identify without a dissecting microscope. Like all small organs they should be wrapped in lens paper to avoid loss during fixation.

Extremities: 

Skeletal muscle: This can be cut from the thigh bones and fixed whole.

Bone marrow: The entire femur from the hip joint to the knee should be cut away from the animal with scissors. All of the muscle should carefully be cut away. Cut off each end of the bone with scissors to reveal the hollow marrow filled core. Bone should be fixed whole in formalin for 24 hours. It then should be transferred to decalcification (11% formic acid with stirring) solution for overnight prior to paraffin processing. One femur should be saved for cytology as well. With a 26G needle flush 1 mL of media (Iscove's +15% heat inactivated serum) through the marrow space and out the other end of the bone into a 1.5 mL tube. Plate 100 µL onto positively charged glass slides with a cytospin centrifuge and stain with Wright-Giemsa. This will preserve the cellular morphology and will allow an accurate differential cell count to be performed.

Head and Neck: 

Mouth: Inspect the mouth for excessive growth of the incisor teeth or abnormal jaw structure. Excessive incisor growth can prevent mice from feeding normally and can cause malnutrition.

Thyroid. The strap muscles connecting running along the neck should be cut away with scissors under a dissecting microscope. When the trachea is well exposed it should be grasped inferiorly with forceps and retracted downwards. It then should be cut above the larynx several mm below to remove it from the neck. The thyroid gland has the same color as muscle and consists of two small patches of tissue adherent to the trachea just below the laryngeal prominence. Under the stereomicroscope it will appear more translucent than muscle. Do not remove the thyroid from the trachea but instead cut across the trachea just above and below the thyroid tissue. For better morphology fix and embed this en bloc and cut cross sections across the trachea.

Eyes: The eyes will protrude slightly if the fur is retracted.   Grasp the orbits with forceps and cut them free from the ocular muscles and optic nerve.   For optimal retinal morphology a sharp slit in the corneas should be made and the lenses removed. To accomplish this make a hole in the cornea with a needle and the cut away the cornea with very fine scissors. Gently remove the lens using fine forceps without distorting the remainder of the eye or detaching the retina. After the corneas and lenses have been removed little hollow cups will remain. Fix these in formalin for paraffin or 5% glutaraldehyde for plastic embedding. Embed the retinal cups on their sides. When sectioning cut the blocks down to the level of the optic nerve for optimal morphology.

Brain: If optimal CNS morphology is required consider infusing fixative intravascularly via intracardiac perfusion prior to dissection. Retract the skin from the skull. The cranial bone should be excised along its perimeter. Start by flexing the neck and inserting the tips of scissors into the foramen magnum at the skull base. Cut the cranium with small snips at its juncture with the facial bones, superiorly to the reveal the entire brain and olfactory lobes. Gently lift the brain under the frontal lobes and free it from underlying vessels and cranial nerves. Remove in one piece the cerebrum, cerebellum and as much of the hind brain as possible. Do not cut the brain until it has been fixed. After paraffin embedding the brain should be cut with a scalpel and embedded cut surface down. At this time you must decide whether you want to view coronal or sagital sections.

Pituitary: After the brain is removed the pituitary will generally remain sitting on the floor of the skull between the two large trigeminal nerves that run longitudinally along the bone. The pituitary is a 2 x 4 mm strip of pale tissue running crosswise between these two nerves. Under the dissecting microscope the thin meningeal membrane that overlies the pituitary should be torn away with forceps. Gently lift out the pituitary with the tip of a curved 26G needle or with fine forceps. Let it sit in a drop of fixative for a few minutes and then wrap it in a small piece of lens paper and transfer it to a tissue cassette for fixation with other small organs. All 3 tissue layers will be visualized if it is embedded upside (posterior) side down or if cut in the coronal plane.

Cleanup:  Although laboratory animals are extremely unlikely to carry human pathogens yet tissues should be treated as potentially infectious and disposed of in biohazardous waste.  Carcasses should be frozen in plastic bags and disposed of the designated location in AHR.   Healthy mice which have been euthanized but were never subjected to any sort of experimental manipulations (e.g. unused wildtype stocks) may be segregated into separate plastic bags and labeled "ZOO" for use as food for reptiles at the local zoo.  Return cages to the cage wash area at AHR but refrain from entering animal housing areas after handling dirty cages to minimize the risk of pathogen transmission.