小鼠的組織學技術--組織處理，染色
Histology of mouse tissues
(M. Fero)
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, 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.
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 very rapid fixative which requires only a few hours. It is routinely used by the U.W. pathology lab for immunohistochemistry, although this is antigen and antibody specific. H&E stains come out much more red in this and any alcohol based fixative.
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.
Dissection
Record the identifying data plus the appearance and behavior of the animal. Record the weight of the animal, 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.
Wet the animals' fur with 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.
Body wall:
Skin: A vertical midline incision with scissors should be cut from the neck to pubis. This should be extended laterally 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 should be dissected out by grabbing 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 MEFs are to be made. This should be pinned back along with the skin.
Uterus and ovary: The pregnant uterus should be obvious by it's "beads on a string" appearance in the lower abdomen. It has two horns which extend up from the cervix in the pelvis. Cut across the cervix and lift out the uterus with forceps. As it is lifted out cut away the fat and mesometrium attachments. It is superiorly on the left and right to an ovary which is situated just below each kidney. For histology the uterus should be fixed whole overnight in formalin and then tranferred to 70% ethanol. It then 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.
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. 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.
Testes: Testes should not be cut prior to fixation because they will rupture. Fix them whole.
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 more easily. It ruptures easily so it should be fixed whole. Prior to embedding it should be cut longitudinally with a scalpel and the cut surface should be embedded face down if possible.
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 and skin infections or ulcers will cause enlargement of the nearby nodes. They are 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 face 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
Femur:
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:
Inspect the mouth for excessive growth of the incisor teeth. This can prevent mice from feeding normally and cause them to starve.
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 grabbed inferiorly with forceps and retracted downwards. It then should be cut above and below and 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 microscope it will appear more translucent than muscle. Leave it attached to the trachea but cut across the trachea just below the thyroid tissue and just above the larynx to leave about a 5 mm section which can be fixed whole. Embed the trachea such that it is cut in cross section. And cut sufficiently deep to visualize both the tracheal cartilage and the adherent thyroid tissue.
Eyes: The eyes will bulge if the fur is retracted. Grab the eyes with forceps and cut them free from the muscle and optic nerve. Holes can be poked in the cornea with a needle to help fixative penetrate. For optimal retinal morphology the lenses should be removed. To do this poke a hole in the cornea with a needle and them cut away the cornea with very fine scissors. Gently remove the lens with forceps without distorting the remainder of the eye or detaching the retina. With the corneas and lenses removed you will have little hollow cups remaining. Fix these in formalin for paraffin or 5% glutaraldehyde for plastic embedding. Embed the eyes on their sides. When sectioning cut the blocks down to the level of the optic nerve for optimal morphology.
Brain: Remove the fur from the head. The cranial bone should be cut away by flexing the animals neck and inserting the tips of bone scissors into the foramen magnum at the base of the skull. Cut the cranium away with small snips along the sides all the way forward to the snout and remove the skull cap. Lift the brain and free it from the vessels and cranial nerves connecting it to the base of the skull. Remove in one piece the cerebrum, cerebellum and as much of the hind brain as possible.
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