Questo lavoro è stato sostenuto dalle sovvenzioni da National Natural Science Foundation of China (31201086, 31470759 e 31671219) e Shanghai Natural Science Foundation (12ZR1436900).

topi C57BL/6 sono allevati in condizioni da organismi patogeni specifiche. Tutti i metodi sperimentali animali sono in conformità con le linee guida approvate dal comitato di etica presso Second Military Medical University e cura degli animali.
 1. dissezione di postnatale sviluppando ghiandola pituitaria <ol><li>eseguire anestesia: posto topi neonatali (P0 - P5) su ghiaccio tritato per indurre ipotermia. Per topi più di 5 giorni di età, iniettare intraperitonealmente 5% uretano (30 µ l/g di peso corporeo) per indurre l'anestesia. Valutare le risposte per coda/tep pizzichi. Procedere solo dopo che il mouse non risponde agli stimoli nocivi.</li>
	<li>Secure il mouse nella posizione supina (sdraiato sulla schiena con la faccia verso l'alto) toccando delicatamente le zampe anteriori e si ad una superficie di lavoro che è capace di essere appuntato (cioè, polistirolo) all'interno di una cappa chimica.</li>
	<li>Eseguire perfusione perfusione.
	<ol><li>Tagliare la gabbia toracica del mouse con forbici chirurgiche per esporre il cuore (e altri organi toracici).</li>
		<li>Fissare il cuore che batte con il forcipe smussato e inserire un ago da 26G (0,45 mm) nel ventricolo di sinistra. L'ago è collegato a una siringa da 10 mL riempita con soluzione fisiologica eparinizzata tampone fosfato (PBS; completati con nitrito di sodio 5 mg/mL e 10 U/mL eparina, pH 7.4, riscaldare a 37 ° C prima dell'uso). Immediatamente tagliare l'atrio destro con forbice e cominciare a irrorare il corpo con il PBS caldo fino a quando il liquido esce l'atrio di destra è chiaro. 
		Nota: Circa 5-10 mL di PBS è necessario per topi neonatali e 10-20 mL di PBS per topi adulti.</li>
		<li>Tenere l'ago in posizione e modificare un'altra siringa riempita con paraformaldeide al 4% (PFA; pH 7.4). Irrorare di 10 mL e 20 mL di fissativo per neonatale e P21 mouse, rispettivamente. 
		Attenzione: PFA è tossico. Può causare danni agli occhi e irritazioni respiratorie e pelle. Indossare guanti e lavorare sotto una cappa chimica.</li>
	</ol></li> <li>Decapitare il mouse PFA-fisso e tagliare l'osso del cranio aperto con le forbici.
	<ol><li>Sollevare delicatamente il hindbrain dalla base del cranio con piccole pinze. Al primo segno del turcica di sella, fermata di sollevamento, ma tenere il hindbrain, tagliare il gambo pituitario e fibre nervose che collega alla base del cervello con forbice. Questo passaggio è fondamentale per garantire che l'ipofisi non viene sollevato con il cervello.</li>
		<li>Quindi mantenere sollevamento cervello e rimuovere l'intero cervello per esporre completamente la ghiandola pituitaria. Tagliare la regione intera sella compreso la ghiandola pituitaria, i nervi di trigeminal laterali e il sotto osso sfenoidale con le forbici (circa 3 x 5 mm 2).</li>
	</ol></li> <li>Mettere il tessuto dissecato in un piatto di 35 mm o un pozzetto di una piastra a 6 pozzetti contenenti 2 mL freddo 4% PFA (pH 7.4). Difficoltà il tessuto a 4 ° C per 40 min per P0 - P7 cadaveri, 1h per P14 cadaveri, 1,5 h per P21 - P28 cadaveri e 3 ore per adulti cadaveri.</li>
	<li>Ulteriore dissociazione del pituitary <ol><li>lavare il tessuto fisso con 10 mL di PBS, 5 cambi, 15 minuti ciascuno. Ghiandola pituitaria neonatale insieme ai relativi tessuti circostanti e il sotto dello sfenoide possono essere elaborati direttamente alla disidratazione. Tuttavia, dovrebbe essere dissociate ghiandole pituitarie dai topi più di 5 giorni più ulteriormente sotto un microscopio stereo.</li>
		<li>Mettere il tessuto fisso in un piatto di 35 mm contenente 1 mL di PBS. Per P5 - P14 cadaveri, rimuovere le membrane connettive tra i nervi e l'osso con una pinzetta e forbici e isolare accuratamente la ghiandola pituitaria insieme ai nervi di trigeminal laterale come un intero, ma lasciando il turcica di sella. La ghiandola isolata e nervi sono collegati da membrane di tessuto connettive con un &#34; H &#34;-l'aspetto ( Figura 1B).</li>
		<li>Per P21 e adulto cadaveri, rimuovere i nervi e tessuto connettive membrane intorno l'ipofisi e gratuito della ghiandola dai tessuti circostanti. Avvolgere il tessuto pituitario con carta velina di pulizia delle lenti e metterlo in una cassetta incasso. 
		Nota: Avvolgimento l'ipofisi piccolo con carta velina di pulizia aiuta a prevenire qualsiasi potenziale perdita di esemplari e preservare l'integrità del tessuto nella seguente procedura inclusione in paraffina. Potrebbe non essere necessario per avvolgere l'ipofisi se viene elaborato in una piccola bottiglia nella procedura cryo-incorporamento.</li>
	</ol></li></ol> 2. Embedding e sezionamento di paraffina <ol><li>disidratare gli esemplari in incorporamento cassette con soluzioni di etanolo di 50%, 65%, 75% e 95% per 15 minuti ciascuno. Successivamente disidratare con 3 cambi di etanolo puro, 3 min ciascuno per P0 - P4 ghiandole, 4 min ciascuno per P5 - P14 ghiandole e 5 minuti ciascuno per P21 e ghiandole più anziani. Trasparente con xilene, 3 cambia, 3 min ciascuno per P0 - P4 ghiandole, 4 min ciascuno per P5 - P14 ghiandole e 5 minuti ciascuno per P21 e ghiandole più anziani. Infiltrarsi con cera di paraffina fusa, 3 cambi, 7 min ciascuno per P0 - P4 ghiandole, e 8 min due volte seguito da 6 min una volta per P5 e più grandi ghiandole. 
	Nota: I parametri ottimali per l'elaborazione del tessuto possono essere regolati empiricamente. Per ottenere sezioni di alta qualità, disidratazione insufficiente o eccedenza dovrebbe essere evitato. Lo stesso vale per la compensazione del tessuto utilizzando xilene.</li>
	<li>Orientamento quando si incorpora <ol><li>su un tessuto che sistema console, rimuovere il campione dal cassetto e inserirlo in uno stampo base riempito a metà con cera di paraffina fusa. Corretto orientamento della ghiandola pituitaria incorporamento è essenziale per soddisfare le sezioni coronali.</li>
		<li>Per P21 e adulto ghiandole pituitarie, posizionare la ghiandola pituitaria con suo breve asse perpendicolare alla superficie di fondo di uno stampo base (il piano di sezione). Utilizzare sfenoide ossa e nervi di trigeminal come limiti anatomici per P0 - P4 e P5 - P14 cadaveri, rispettivamente. Orientare gli esemplari con i loro nervi di trigeminal o lamina trasversale delle ossa sfenoide perpendicolare alla superficie di fondo di uno stampo base.</li>
		<li>Premere delicatamente il tessuto nella posizione desiderata con una pinzetta riscaldata fino a che la cera diventa semi-solida su una piastra di raffreddamento. Riempire lo stampo con cera di paraffina fusa. Consentire il blocco di paraffina raffreddare fino a quando la cera è completamente solidificata. 
		Nota: L'ipofisi al giorno embrionale 18,5 (E18.5) può essere trattata nello stesso modo come l'ipofisi neonatale. Ma i cadaveri (Rathke &#39; sacchetti s) più giovane di E16.5 dovrebbe essere incorporato con l'intera testa e orientato con l'asse sagittale (dalla bocca all'occipite) perpendicolare alla superficie di fondo di uno stampo base.</li>
	</ol></li> <li>Chill il blocco di paraffina a-20 ° C per 10-20 min e poi tagliati in sottili fette (4 µm spessore è preferibile) con un microtomo. Per ottenere sezioni coronali soddisfacente, mettere a punto la posizione dei blocchi di paraffina durante il sezionamento. Montare le fette di tessuto su vetrini polilisina-rivestito per evitare il distacco delle fette nelle procedure seguenti. 
	Nota: In alternativa, gli esemplari possono essere disidratati in 15%, 30% (P/V) soluzioni di saccarosio (in PBS) a 4 ° C, finché non si depositano sul fondo, incorporato in cryo-incorporamento composto utilizzando lo stesso metodo di orientamento e rapidamente congelato in ghiaccio secco. Quindi tagliare i blocchi di tessuto congelato fette di 8-10 µm utilizzando un criostato. La morfologia del cryo-sezioni, tuttavia, è spesso inferiore a sezioni di paraffina.</li>
</ol> 3. H &#38; E macchiatura e immunofluorescenza etichettatura <ol><li>caldo le diapositive a fuoco bloccano a 55 ° C e Sparaffinatura con xilene, 2 cambi, 4 min. Disidratare i campioni con etanolo al 95% due volte e due volte, etanolo di 75% 3 min ogni. Lavare i vetrini con acqua distillata per 5 minuti su un agitatore.</li>
	<li>Per H &#38; E colorazione, macchia le sezioni in soluzione di ematossilina di allume per 6 min, sciacquare in runnl'acqua del rubinetto ing per 2 min, differenziare con acqua ammoniaca 0,2% per 45 s e ri-sciacquare in acqua corrente per 2 min. Quindi disidratare le sezioni con etanolo al 95% per 1 min e macchia con eosina per 2 min in sequenza disidratare, chiaro e montare.</li>
	<li>Immunofluorescenza etichettatura <ol><li>trattare le sezioni con metanolo contenente 3% H 2 O 2 e 0,01% NaN 3 per 20 min a temperatura ambiente per inattivare le perossidasi endogene. Recuperare gli antigeni bollendo sezioni nel buffer di recupero (1 mM EDTA e 1 mM Tris-HCl, pH 7.4) in una pentola a pressione per 2 min. Incubare le sezioni con tampone bloccante (5% di siero di capra in PBS) per 30 min a 37 ° C.</li>
		<li>Incubare sezioni con anticorpo di coniglio anti-somatotropina (GH) (1:2, 000) o anticorpi di coniglio anti-proteina fibrillare acida (GFAP) (1:2, 000) diluiti in tampone bloccante durante la notte a 4 ° C.</li>
		<li>Incubare sezioni con anticorpo secondario (capra anti-coniglio IgG-HRP, 1: 300 in tampone bloccante) 35 min a 37 ° C.</li>
		<li>Amplificare i segnali con Biotinyl Tyramide (01:50 in soluzione salina tamponata Tris contenente 0,3% H 2 O 2) per 15 min a temperatura ambiente e visualizzare con streptavidina-Alexa594 o 488 (1: 300 in blocco buffer, 30 min, 37 ° C).</li>
		<li>Colorante di contrasto i nuclei con DAPI (50 mg/L in PBS) per 5 min a temperatura ambiente. 
		Nota: Potrebbe non essere necessario amplificare il segnale usando il sistema di amplificazione del segnale di microarray (TSA). In alternativa, un anticorpo secondario coniugato a fluorescenza può essere utilizzato per visualizzare il segnale.</li>
	</ol></li> <li>Acquisire immagini utilizzando un microscopio a fluorescenza dotato di DAPI, Texas Red e set di filtri FITC, × 4/0,13 a × 40/0.75 obiettivi e fotocamera da 5.0 megapixel. Utilizzare il 360, 488 e 594 lunghezze d'onda laser nm per imaging DAPI, Alexa 488 - e sezioni macchiate di Alexa 594, rispettivamente. Ottimizzare la potenza del laser (0.8 è stato utilizzato generalmente) e tempo di esposizione ai livelli desiderati per ogni canale. Acquisire l'immagine sotto il controllo del software di acquisizione immagini e sovrapporre le immagini di fluorescenza successivamente utilizzando software di elaborazione immagine.</li>
</ol>

<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. 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Per lo sviluppo di cadaveri murini, è stato tecnicamente difficile ottenere sezioni coronali corretto a causa della loro piccole e fragile caratteristiche e peculiarità anatomiche6,8. Alcuni gruppi di ricerca ha così scelto sagittale sezioni per analizzare la morfologia di embrionale e neonatale pituitario11,12. Però la sezione sagittale del pituitary è anche capace di risultati intermedi, anteriore ...

La ghiandola pituitaria o ipofisi sono un importante organo endocrino che secerne ormoni essenziali per l'omeostasi1,2. Anatomicamente, la ghiandola pituitaria è una struttura di ' due-in-one ' costituita neurohypophysis e adenohypophysis. Queste parti hanno diverse origini embrionali e funzione in modo molto diverso. Neurohypophysis è derivato da ectoderma neurale e secerne l'ormone antidiuretico e l'ossitocina. Adenohypophysis proviene da di Rathke ed è responsabile per il rilascio degli ormoni tra cui l'ormone della crescita, prolattina, ormone stimolante la tiroide, ormone follicolo - stimolante, ormone luteinizzante, ormone adrenocorticotropo, e ormone distimolazione3,4,5.
La ghiandola pituitaria si riposa sulla superficie dorsale dell'osso sfenoidale (sella turcica) del cranio del mouse e viene fissata al pavimento del cervello da un gambo fragile. Lateralmente è circondato dai nervi di trigeminal e anteriormente dal chiasma ottico6,7. La ghiandola ha una forma ovale allungata con il suo asse perpendicolare a quella della testa. Sulla sua superficie dorsale, neurohypophysis e adenohypophysis può facilmente essere delimitate, con l'ex che occupa la regione dorsale mediale e il quest'ultimo che si estende lateralmente e ventralmente. Durante lo sviluppo postnatale, la dimensione della ghiandola pituitaria aumenta rapidamente nel primo mese dopo la nascita8,9. Tuttavia, la ghiandola pituitaria del mouse è ancora molto di piccole dimensioni con un diametro assi lunghi di ~ 3 mm e un peso medio di 1,9 mg in adulti10, diametro assi lunghi 2-2,5 mm al giorno postnatale 21 (P21) e solo l'1-1,5 mm in P0.
Una sezione coronale è preferita per visualizzare sia adenohypophysis e neurohypophysis in una sola fetta del pituitary del mouse. Tuttavia, alcune abilità tecniche sono necessario per ottenere soddisfazione sezioni coronali di ghiandole pituitarie da topi grazie alle sue dimensioni estremamente ridotte e distinta anatomia di sviluppo. In questo articolo dei video, dimostriamo come sezionare ghiandole pituitarie del mouse e preparare sezioni coronali pituitari alle diverse fasi di sviluppo.

<table><tbody><tr><td>&lt;strong&gt;Tools/Equipment&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</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&amp;amp;E staining</td></tr><tr><td>Eosin Y</td><td>Sigma</td><td>E4009</td><td>for H&amp;amp;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

La ghiandola pituitaria o ipofisi sono un importante organo endocrino che secerne ormoni essenziali per l'omeostasi. Si compone di due ghiandole con origini embrionali separate e funzioni — neurohypophysis e adenohypophysis. Ghiandola pituitaria del mouse in via di sviluppo è molto piccolo e delicato con una forma ovale allungata. Una sezione coronale è preferita per visualizzare sia il adenohypophysis e neurohypophysis in una sola fetta del pituitary del mouse.
L'obiettivo del presente protocollo è quello di ottenere sezioni coronali pituitari corretto con le architetture di tessuto ben conservato da topi di sviluppo. In questo protocollo, descriviamo dettagliatamente come sezionare ed elaborare ghiandole pituitarie correttamente da topi di sviluppo. In primo luogo, topi vengono fissati mediante perfusione perfusione di formaldeide prima della dissezione. Quindi tre diverse tecniche di dissezione vengono applicate per ottenere ghiandole pituitarie intatti a seconda dell'età dei topi. Per topi fetali dai giorni embrionale (E) 17,5-18,5 e neonati fino a 4 giorni, le regioni di sella intera compreso l'osso sfenoidale, ghiandola e nervi di trigeminal vengono sezionate. Per i cuccioli invecchiati giorni postnatali (P) 5-14, ghiandole ipofisi collegati con nervi di trigeminal vengono sezionati nel suo complesso. Per i topi vecchio oltre 3 settimane, le ghiandole pituitaria attentamente vengono sezionate gratis dai tessuti circostanti. Visualizziamo anche come incorporare le ghiandole pituitaria in un orientamento corretto utilizzando i tessuti circostanti come punti di riferimento per ottenere soddisfazione sezioni coronali. Questi metodi sono utili per analizzare le caratteristiche istologiche e dello sviluppo delle ghiandole pituitaria in topi di sviluppo.

Questo protocollo presenta un metodo per sezionare ghiandole pituitarie da topi di sviluppo. Per il mouse neonatale, le regioni di sella intera che contiene la ghiandola pituitaria, i nervi di trigeminal e la sottostante osso sfenoidale sono stati sezionati fuori dalla base del cranio. La ghiandola pituitaria piccola e delicata rimasto intatta durante il processo (Figura 1A). Per topi più di 5 giorni, le ghiandole pituitaria collegate ai nervi di trigeminal ...

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

Dissezione e preparazione coronale fetta di sviluppare la ghiandola pituitaria del Mouse

Vi presentiamo un protocollo per sezionare le ghiandole pituitarie e preparare sezioni coronali pituitarie da topi di sviluppo.

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

dissection-coronal-slice-preparation-developing-mouse-pituitary

Research

JoVE Journal

Developmental Biology

17.3K Views.  Second Military Medical University. Vi presentiamo un protocollo per sezionare le ghiandole pituitarie e preparare sezioni coronali pituitarie da topi di sviluppo.

Video: Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Un approccio per la valutazione optogenetic Formazione di connessioni neuronali in un sistema di co-coltura

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Ricerca

Biologia dello sviluppo

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Un saggio Slice organotipica per High-Resolution Time-Lapse Imaging della migrazione neuronale nel cervello postnatale

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Neuroscienze

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Preparazione di Synaptoneurosomes da mouse Cortex con un discontinuo di Percoll-Saccarosio gradiente di densità

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Preparazione di sezioni parasagittali delle inchieste sui dorso-ventrale organizzazione della corteccia entorinale Rodent mediale

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

Modifica di un Colliculo-talamo-corticale mouse Cervello Fetta, Incorporando stampa 3-D della Camera componenti e multi-scala Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Un tumore cerebrale / organotipica fetta sistema di co-coltura per lo Studio microambiente tumorale e mirata terapie farmacologiche

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Medicina

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

Isolamento ed espansione delle neurosfere dalle nicchie neurogeniche del topo postnatali (P1-3)

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

Microdissezione del cervello di topo in regioni funzionalmente e anatomicamente diverse

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.