This work was supported by the grants from National Natural Science Foundation of China (31201086, 31470759 and 31671219) and Shanghai Natural Science Foundation (12ZR1436900).

C57BL/6 mice are bred in specific pathogen-free conditions. All animal experimental methods are in compliance with the guidelines approved by the Animal Care and Ethics Committee at Second Military Medical University.
1. Dissection of Postnatal Developing Pituitary Gland
<ol>
	<li>Perform anesthesia: Place neonatal mice (P0 - P5) on crushed ice to induce hypothermia. For mice older than 5 days of age, inject 5% urethane (30 &#181;L/g of body weight) intraperitoneally to induce anesthesia. As...

<ol>
	<li>Fauquier, T., Lacampagne, A., Travo, P., Bauer, K., Mollard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+face+of+the+anterior+pituitary.">Hidden face of the anterior pituitary.</a> Trends Endocrinol Metab. 13 (7), 304-309 (2002).</li><li>Haedo, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pituitary+function+by+cytokines.">Regulation of pituitary function by cytokines.</a> Horm Res. 72 (5), 266-274 (2009).</li><li>Zhu, X., Gleiberman, A. S., Rosenfeld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+pituitary+development:+signaling+and+transcriptional+networks.">Molecular physiology of pituitary development: signaling and transcriptional networks.</a> Physiol Rev. 87 (3), 933-963 (2007).</li><li>Bancalari, R. E., Gregory, L. C., McCabe, M. J., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+gland+development:+an+update.">Pituitary gland development: an update.</a> Endocr Dev. 23, 1-15 (2012).</li><li>Kelberman, D., Rizzoti, K., Lovell-Badge, R., Robinson, I. C., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+pituitary+gland+development+in+human+and+mouse.">Genetic regulation of pituitary gland development in human and mouse.</a> Endocr Rev. 30 (7), 790-829 (2009).</li><li>Peker, S., Kurtkaya-Yapicier, O., Kilic, T., Pamir, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+anatomy+of+the+lateral+walls+of+the+pituitary+fossa.">Microsurgical anatomy of the lateral walls of the pituitary fossa.</a> Acta Neurochir (Wien). 147 (6), 641-648 (2005).</li><li>Wolpert, S. M., Molitch, M. E., Goldman, J. A., Wood, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size,+shape,+and+appearance+of+the+normal+female+pituitary+gland.">Size, shape, and appearance of the normal female pituitary gland.</a> AJR Am J Roentgenol. 143 (2), 377-381 (1984).</li><li>Carbajo-Perez, E., Watanabe, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+proliferation+in+the+anterior+pituitary+of+the+rat+during+the+postnatal+period.">Cellular proliferation in the anterior pituitary of the rat during the postnatal period.</a> Cell Tissue Res. 261 (2), 333-338 (1990).</li><li>Nantie, L. B., Himes, A. D., Getz, D. R., Raetzman, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+postnatal+pituitary+expansion:+proliferation,+progenitors,+and+cell+specification.">Notch signaling in postnatal pituitary expansion: proliferation, progenitors, and cell specification.</a> Mol Endocrinol. 28 (5), 731-744 (2014).</li><li>Tzou, S. C., Landek-Salgado, M. A., Kimura, H., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+pituitary+immunogen+for+the+induction+of+experimental+autoimmune+hypophysitis.">Preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis.</a> J Vis Exp. (46), (2010).</li><li>Budry, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Related+pituitary+cell+lineages+develop+into+interdigitated+3D+cell+networks.">Related pituitary cell lineages develop into interdigitated 3D cell networks.</a> Proc Natl Acad Sci U S A. 108 (30), 12515-12520 (2011).</li><li>Wilson, D. B., Wyatt, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+the+pituitary+gland+in+neonatal+little+(lit)+mutant+mice.">Histopathology of the pituitary gland in neonatal little (lit) mutant mice.</a> Histol Histopathol. 7 (3), 451-455 (1992).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> J Neurosci Methods. 12 (2), 141-149 (1984).</li><li>Cao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZBTB20+is+required+for+anterior+pituitary+development+and+lactotrope+specification.">ZBTB20 is required for anterior pituitary development and lactotrope specification.</a> Nat Commun. 7, 11121 (2016).</li></ol>

The authors have nothing to disclose.

For developing murine pituitaries, it has been technically difficult to obtain proper coronal sections due to their tiny and fragile features and unique anatomical characteristics6,8. Some research groups thus chose mid-sagittal sections to analyze the morphology of embryonic and neonatal pituitary11,12. Though the mid-sagittal section of pituitary is also capable of showing anterior, intermediate, and po...

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis1,2. Anatomically, the pituitary gland is a &#39;&#39;two-in-one&#39;&#39; structure consisting of the neurohypophysis and the adenohypophysis. These parts have different embryonic origins and function very differently. The neurohypophysis is derived from the neural ectoderm and secretes oxytocin and antidiuretic hormone. The adenohypophysis originates from Rathke&#39;s pouch and is responsible for the release of hormones including growth hormone, prolactin, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, and melanocyte-stimulating hormone3,4,5.
The pituitary gland rests on the dorsal surface of the sphenoid bone (sella turcica) of the mouse skull and is attached to the floor of the brain by a fragile stalk. It is surrounded laterally by trigeminal nerves and anteriorly by the optic chiasm6,7. The gland has an elongated oval shape with its long axis perpendicular to that of the head. On its dorsal surface, the neurohypophysis and the adenohypophysis can easily be demarcated, with the former occupying the dorsal medial region and the latter extending laterally and ventrally. During postnatal development, the size of pituitary increases rapidly in the first month after birth8,9. Nevertheless, the mouse pituitary is still very small in size with an average weight of 1.9 mg and a long-axis diameter of ~3 mm in adult10, a long-axis diameter of 2 - 2.5 mm at postnatal day 21 (P21), and only 1 - 1.5 mm at P0.
A coronal section is preferred to display both adenohypophysis and neurohypophysis in a single slice of the mouse pituitary. However, some technical skills are required to obtain satisfying coronal sections of pituitary glands from developing mice due to its exceptionally small size and distinct anatomy. In this video article, we demonstrate how to dissect mouse pituitary glands and prepare pituitary coronal sections at different development stages.

<table>
	<tbody>
		<tr>
			<td>Tools/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors-straight</td>
			<td>JinZhong</td>
			<td>J21010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine scissors-strainght</td>
			<td>JinZhong</td>
			<td>WA1010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>JinZhong</td>
			<td>JD1020</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>RS-5015</td>
			<td>for isolation of the pituitary</td>
		</tr>
		<tr>
			<td>26G (0.45mm) needle</td>
			<td>HongDa</td>
			<td></td>
			<td>for transcardial perfusion</td>
		</tr>
		<tr>
			<td>Syringe (1 mL)</td>
			<td>BD</td>
			<td>300841</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Syringe (10 mL)</td>
			<td>BD</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Lens cleaning paper</td>
			<td>ShuangQuan</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Anatomical microscope</td>
			<td>OLYMPUS</td>
			<td>SZX-ILLB2-200</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Embedding cassette</td>
			<td>Thermo Fisher</td>
			<td>22-272423</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Tissue embedding console system</td>
			<td>KEDEE</td>
			<td>KD-BM11</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo Fisher</td>
			<td>HM315R</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Superfrost-Plus slides</td>
			<td>Thermo Fisher</td>
			<td>22-037-246</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Thermo Fisher</td>
			<td>12-543</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>BH2-RFCA</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>BBI</td>
			<td>EB0448</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4.12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P2222</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>NaNO2</td>
			<td>Sigma</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection</td>
			<td>SPH</td>
			<td>H31022051</td>
			<td>for perfusion saline</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SCR</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SCR</td>
			<td>10023418</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Thermo Fisher</td>
			<td>8330</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma</td>
			<td>H9627</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>E4009</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>rabbit anti growth hormone (GH)</td>
			<td>National Hormone</td>
			<td></td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>antibody</td>
			<td>Pituitary Program</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse GFAP antibody</td>
			<td>Sigma</td>
			<td>G9269</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, HRP</td>
			<td>Jackson</td>
			<td>111-035-003</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>TSA system</td>
			<td>NEN Life Science Products</td>
			<td>NEL700</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 594</td>
			<td>Thermo Fisher</td>
			<td>S32356</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Anti-FITC Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A11090</td>
			<td>for immunostaining</td>
		</tr>
	</tbody>
</table>

dissection and coronal slice preparation of developing mouse pituitary gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

This protocol presents a method to dissect pituitary glands from developing mice. For the neonatal mouse, the whole sella regions containing the pituitary gland, the trigeminal nerves, and the underneath sphenoid bone were dissected out from the skull base. The tiny and delicate pituitary gland remained intact during the process (Figure 1A). For mice older than 5 days, the pituitary glands attached to the lateral trigeminal nerves were then isolated. The gros...

Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

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.

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Research

JoVE Journal

Developmental Biology

16.8K Views.  Second Military Medical University. 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 ha...