The overall goal of this procedure is to assess mammary gland development in wild type and mutant mice. This is accomplished by first dissecting a mouse to expose the mammary glands. Next, the mammary glands are properly harvested and spread on glass slides, calm lum.
Staining is then used to analyze the extent of epithelium development. Finally, analysis is carried out of myoepithelial contractions upon oxytocin exposure, INEA J.Ultimately, the results obtained will reveal if the mammary gland ducts and the alveoli have properly formed, and whether the myo epithelium can be induced to contract and eject milk upon oxytocin exposure. This method can help answer key questions related to mammary gland development and differentiation, such as the impact of gene mutation or knockout on mammary gland morphology and function.
Visual demonstration of this method ensures proper dissection of the mammary gland as both hole mounting and the oxytocin based assay require well-prepared mammary glands. Poor dissection of the gland may result in the absence of some epithelial ducts or the presence of extraneous tissues such as skin or muscle tissue At least one day prior to dissection. Prepare CalMin alum solution by dissolving one gram of CalMin and 2.5 grams of aluminum potassium sulfate in 500 milliliters of distilled water.
Boil the solution for 20 minutes in a one liter flask, adjust the final volume to 500 milliliters with water. Filter the solution through a watman paper and add a few grains of thiol as a preservative. Then refrigerate the solution after staining.
Carbon alum solution can be poured back into the stock bottle and reused many times over several weeks. Make a fresh solution when the color becomes faint. To begin memory gland dissection, sacrifice a mouse using carbon dioxide inhalation.
Avoid cervical dislocation if possible since it may damage major blood vessels in the neck and result in the accumulation of blood around mammary gland. Number one, rendering its dissection more difficult on polystyrene foam wrapped in foil. Place the mouse on its back and spread the four limbs.
Pin firmly all four limbs using 16 to 20 gauge needles. Spray the mouse generously with 70%ethanol using forceps. Pull up the abdominal skin at the midline and make a small incision with sharp scissors starting from the incision.
Cut the skin up to the neck of the animal while avoiding puncturing the abdominal or thoracic cavities. Cut the skin on the front legs to the midline incision resulting in a Y shape. Cut the skin on the rear limbs the same way.
Using hemostatic forceps. Gently peel the abdominal and thoracic skin to the side of the mouse if needed. Gently cut the conjunctive tissue.
Fix the skin to the polystyrene foam using 16 to 20 gauge needles, exposing the memory glands attached to the underside of the skin. Dissect one gland at a time using forceps. Gently lift the mammary gland of interest and cut between the gland and the skin with small sharp scissors starting from the outside towards the spine of the animal.
Be sure to cut carefully so no pieces of skin or muscle remain on the gland. All mammary glands can be removed using this protocol, but abdominal mammary glands number four are best for whole mounting. After excising the mammary gland from the mouse, spread it directly on a glass slide.
Try to flatten it as much as possible. Keeping the original in situ shape, the gland will stick on the slide nicely. When properly stretched in a fume hood, fix the gland by immersing the slide into a jar of car noise.
Fixative for four hours at room temperature. Alternatively, fix the gland overnight at four degrees Celsius. Next, wash the tissue in 70%ethanol for 15 minutes.
Then rehydrate it gradually in water by removing half of the ethanol solution from the jar and replacing it with double distilled water. Incubate for five minutes. Repeat three times, rinse and dis distilled water for five minutes.
Then stain the gland in carbon alum overnight at room temperature the next day, remove the carbon alum stain to reuse and gradually dehydrate the stained tissue through serial ethanol baths for five minutes each. Then clear the tissue in xylene overnight after clearing, immerse the mammary gland in methyl salicylate, keeping methyl salicylate in a fume hood until images are ready to be taken. Mammary gland development can be quantified by counting the number of ramifications or side branches along portions of ducts, the length of the primary ducts or a ratio of epithelial to adipose tissue area could be assessed.
A second essay used to evaluate mammary gland development and function involves exposing the gland to oxytocin and assessing milk ejection into the epithelial ducts. To perform this assay, freshly fed pups should be weaned from the mother and the mother kept in isolation for one hour. Next, sacrifice the mother and expose the mammary glands as described for the mammary gland dissection.
However, leave them in the animal. Begin the assay by using a transfer pipette to evenly drop three to five milliliters of either PBS or the oxytocin solution directly onto the mammary gland of interest. For this procedure, it is recommended to use the thoracic glands using one side as a control and the other side to test milk ejection.
Incubate the glands for one minute and remove the solutions by carefully aspirating them with a transfer pipette monitor milk entry into the ducts by videotape recording or by taking images before and after PBS or oxytocin exposure. Here are examples of mouse memory. Gland hole mounts epithelium ducts indicated by arrows, and the lymph node marked by an arrow head are easily observed in a nicely spread whole mammary gland.
A poorly spread mammary gland may become detached from the slide and collapse rendering its unusable. An improperly excised mammary gland can either have missing parts or pieces of muscle or skin remaining here. Oxytocin induced milk ejection can be observed in epithelium ducks following oxytocin exposure.
However, if ducks are already filled with milk at the beginning of the assay due to an inappropriately long period of latency after the last suckling of the pups, further accumulation of milk in the ducks after oxytocin exposure is hard to observe. Alternatively, a short latency period after pups suckling will prevent the accumulation of enough milk into the alveoli to be properly observed in the ducts after oxytocin exposure Following dissection of mammary glands. Instead of hole mounting or assessing oxytocin induced milk ejection, the mammary glands can be frozen, cryo sectioned, and immuno labeled for any number of resident proteins at the mamm glands.
Or alternatively, the glands can be lyed and prepared for western blotting. Together those two assess allowed for fundamental questions related to gland development, differentiation and function to be addressed in any number of mouse model that have been engineered to lack key genes or arbor genes mutations.
