Dissectie en coronale segment voorbereiding van de ontwikkeling van de hypofyse muis

The overall goal of this procedure is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. This procedure can help answer key questions in the field of pituitary development, such as pituitary histology, cell differentiation, proliferation, and apoptosis. The main advantage of this technique is that developing murine pituitaries can be dissected without visible damage and properly oriented for achieving coronal sections.

We first had the idea for this method when we found that, for developing murine pituitaries, it was technically difficult to obtain proper coronal sections. After anesthetizing and fixing mice according to the text protocol, use scissors to cut the skull bone open. Next, with forceps, gently lift the hindbrain from the base of the skull.

Then, at the first sign of the sella turcica, stop lifting but hold the hindbrain, and use fine scissors to cut the pituitary stalk and nerve fibers connected to the base of the brain. Continue to lift and remove the whole brain to fully expose the pituitary gland. The pituitary gland rests on the dorsal surface of the sphenoid bone and is surrounded laterally by trigeminal nerves.

Then use scissors to cut the entire sellar region including the pituitary gland, lateral trigeminal nerves, and beneath the sphenoid bone. Put the tissue into a 35 mm dish containing 4%PFA, and incubate it at 4 degrees Celsius for 40 minutes to 3 hours, depending on the ages. Then use 10 mL of PBS to wash the fixed tissue for five changes at 15 minutes each.

Transfer the fixed tissue into a 35 mm dish containing 1 mL of PBS, and under a stereo microscope, dissociate the pituitary glands. For P5 to P14 pituitaries, with fine forceps and scissors remove the connective membranes between the nerves and the bone, and carefully isolate the pituitary gland together with lateral trigeminal nerves as a whole, but leaving the sella turcica. For P21 and adult pituitaries, remove the nerves and connective membranes around the pituitary, and free the gland from the surrounding tissues.

After dehydration, xylene clearing, and wax infiltration carried out according to the text protocol, on a tissue embedding console system, remove the specimen from the cassette and place it into a base mold half-filled with molten paraffin wax. For P21 and adult pituitary glands, use warmed fine forceps to position the pituitary gland with its short axis perpendicular to the bottom surface of a base mold. For P0 to P4 pituitaries, orient the specimens with their sphenoid bones perpendicular to the bottom surface of a base mold.

For P5 to P14 pituitary glands, orient the specimens with their trigeminal nerves perpendicular to the bottom surface of a base mold. After positioning the gland, use warmed fine forceps to gently hold the tissue in the desired position until the wax becomes semi-solid on a cooling plate. Top up the mold with molten paraffin wax.

Allow the paraffin block to cool until the wax is fully solidified. Chill the paraffin block at minus 20 degrees Celsius for 10 to 20 minutes, then use a microtome to cut it into thin slices, fine tuning the position of the paraffin block during sectioning. Carry out immunolabeling according to the text protocol.

As shown here, to dissect the pituitary gland from the neonatal mouse, the whole sellar region containing the pituitary gland, the trigeminal nerves, and the sphenoid bone underneath were dissected out from the skull base. The tiny and delicate pituitary gland remained intact during the process. For mice older than five days, the pituitary glands attached to the lateral trigeminal nerves were isolated.

The growth structure of the pituitary gland isolated from the P7 mouse was well-preserved. Here, the pituitary gland of the P21 mouse was successfully isolated without visible damage while removing its surrounding tissue. In this figure, H&E staining of properly-oriented coronal sections demonstrates well-preserved morphology of both adenohypophysis and neurohypophysis in P0, P7, and P21 pituitary glands.

Finally, the processed slides were also compatible with immunofluorescence labeling. As an example, the adenohypophysis and neurohypophysis showed specific immunolabeling of GH and GFAP, respectively. Once mastered, the process from dissection to embedding can be done in 7.5 hours in adult mice and even less in younger mice if it's performed properly.

While attempting this procedure, it's important to remember to dissect the entire sellar region to avoid touching pituitary gland with any surgical tools. Following this procedure, the pituitary that has not been prefixed can also be isolated. In that case, more caution should be taken to avoid any undesired damage.

The gland should also be dissected as quickly as possible. After watching this video, you should have a good understanding of how to dissect mouse pituitary glands and prepare pituitary coronal sections at different developmental stages.