Hiroki Ueharu, Saishu Yoshida, Takako Kikkawa, Naoko Kanno, Masashi Higuchi, Takako Kato, Noriko Osumi, & Yukio Kato. Gene tracing analysis reveals the contribution of neural crest-derived cells in pituitary development. Journal of Anatomy. First published: 27 December 2016. DOI: 10.1111/joa.1257

...Immunohistochemistry

Immunohistochemistry was performed as described previously (Yoshida et al. 2009, 2015). The following primary antibodies were used: chicken IgY against jelly fish GFP (1 : 500 dilution; Aves Labs, Tigard, OR, USA); rabbit IgG against Cre recombinase (1 : 150 dilution; CST, Danvers, MA, USA); goat IgG against human SOX2 (1 : 400 dilution; Neuromics, Edina, MN, USA); guinea pig IgG against rat PROP1 [5 ng μL−1; generated in-house (Yoshida et al. 2009)]; and rabbit IgG against rat NG2 (1 : 400 dilution; Millipore, Darmstadt, Germany). Antisera against anterior pituitary hormones included guinea pig antisera against human growth hormone (GH; 1 : 1500 dilution), which was kindly provided by Dr S. Tanaka (Shizuoka University, Shizuoka, Japan), and rabbit antisera against rat prolactin (PRL; 1 : 5000 dilution), which was kindly provided by Dr Wakabayashi (Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan). Guinea pig antisera against rat adrenocorticotropic hormone (ACTH; 1 : 10000 dilution), rat thyroid-stimulating hormone (TSH)β (1 : 50000 dilution), rat luteinizing hormone (LH)β (1 : 3000 dilution) and rat follicle-stimulating hormone (FSH)β (1 : 3000 dilution) were kindly provided by Dr A. F. Parlow (National Hormone and Pituitary Program, Torrance, CA, USA). A cocktail of antisera against six pituitary hormones was prepared by mixing each antiserum in appropriate ratios, as described above. Secondary antibodies used were Cy3-, Cy5-, or fluorescein isothiocyanate (FITC)-conjugated AffiniPure donkey IgG anti-chicken IgY or anti-rabbit, anti-goat and anti-guinea pig IgG (1 : 500 dilution; Jackson ImmunoResearch, West Grove, PA, USA). Finally, the sections were enclosed in VECTASHIELD mounting medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector, Burlingame, CA, USA). Immunofluorescence was observed under a BZX-700 fluorescence microscope (Keyence, Osaka, Japan).

Cell numbers positive for GFP, individual hormones, SOX2 and DAPI were counted for each section (6500–9500 cells per section) prepared from three male mice (P60).....

Andrew Tai, Martin Cheung, Yong-Heng Huang, Ralf Jauch, Marianne E. Bronner & Kathryn S. E. Cheah. SOXE neofunctionalization and elaboration of the neural crest during chordate evolution. Scientific Reports 6, Article number: 34964 (2016) doi:10.1038/srep34964

...In situ hybridisation and immunohistochemistry

Transfected embryos were fixed for an hour at 4 °C in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), cryoprotected with 30% sucrose in PB and cryosectioned. In situ hybridization on cryosections was performed as described13, using probes for chick, Sox1052, Wnt3a (a gift from N. Itasaki) and FoxD3 chick expressed sequence tag clone. Immunohistochemical staining was performed using the antibodies against: green fluorescent protein (GFP) (Molecular Probes), Laminin (Sigma), HNK-1 (Becton Dickinson), HuC/D (Invitrogen), SOX2 (Neuromics), Islet1/2 (DSHB), P0 (DSHB) and MelEM (DSHB). Immunofluorescence images were photographed using a Zeiss LSM700 confocal microscope in the Faculty Core Facility, Li Ka Shing Faculty of Medicine, the University of Hong Kong....

Kotaro Horiguchi, Hideji Yako, Saishu Yoshida, Ken Fujiwara, Takehiro Tsukada, Naoko Kanno, Hiroki Ueharu, Hiroto Nishihara, Takako Kato, Takashi Yashiro, Yukio Kato. S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland. PLOS One. October 3, 2016http://dx.doi.org/10.1371/journal.pone.0163981. 

...Immunohistochemistry

Heads of S100β/GFP-TG embryonic rats on E15.5, E18.5, E19.5, and E20.5 were fixed in 4% paraformaldehyde buffered with 0.05 M phosphate buffer (PB; pH 7.4) for 20-24 h at 4°C, followed by immersion for more than two days in PB (pH 7.2) containing 30% sucrose at 4°C. This was followed by embedding the samples in optimum cutting temperature (O.C.T.) compound (Sakura Finetek Japan, Tokyo, Japan) at -80°C before sectioning. Frozen sections 10 μm thick in the sagittal plane were mounted on glass slides (Matsunami, Osaka, Japan). Antigen retrieval was performed with an Immunosaver (Nisshin EM, Tokyo, Japan) for 60 min at 80°C. Slides were then washed in 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (pH 7.5) containing 100 mM NaCl (HEPES), followed by blocking in HEPES containing 0.4% Triton X100 and 10% fetal bovine serum (FBS) or 1% bovine serum albumin (BSA). Primary antibodies, antisera, and lectin were reacted overnight at 4°C. The primary antibodies used [...] goat IgG against stem/progenitor cell marker human SOX2 (1:500 dilution, Neuromics, Edina, MN, USA), [...] Afterwards, a cocktail of the following guinea pig antisera against pituitary hormones was used: anti-rat LHβ (1:8,000 dilution), anti-rat FSHβ (1:4,000 dilution), anti-rat TSHβ (1:16,000 dilution), and anti-rat PRL (1:4,000 dilution). The antisera were provided by the National Institute of Diabetes and Digestive and Kidney Disease, courtesy of Dr. A. F. Parlow. Guinea pig anti-human ACTH (1:8,000 dilution) and anti-human GH (1:1,000 dilution) antisera were provided by Dr. S. Tanaka, Shizuoka University, and isolectin B4-conjugated Dyelight 649 (1:100 dilution) was provided by Vector Laboratories (Burlingame, CA, USA). Sections were washed with HEPES, followed by incubation with secondary antibodies, performed with Cy3- or Cy5-conjugated AffiniPure donkey with anti-goat, -mouse, -rabbit, and -guinea pig IgG and fluorescein isothiocyanate (FITC)-conjugated AffiniPure donkey with anti-chicken IgY (1:500 dilution, Jackson ImmunoResearch, West Grove, PA, USA). The sections were again washed with HEPES and then enclosed in VECTASHIELD Mounting Medium with 4',6-diamino-2-phenylindole (DAPI; Vector Laboratories) to stain nuclei. Immunofluorescence was observed under fluorescence microscopy with a BZ-9000 (KEYENCE, Osaka, Japan) and fluorescence confocal microscopy with a FV1000 (OLYMPUS, Tokyo, Japan)....

Elisa Garbayoa, Eduardo Ansorenab, Hugo Lanaa, María del Mar Carmona-Abelland, Irene Marcillae, José Luis Lanciegob, María Rosario Luquinb, María José. Blanco-Prietoa. Brain delivery of microencapsulated GDNF induces functional and structural recovery in parkinsonian monkeys. doi.org/10.1016/j.biomaterials.2016.09.015.

...goat α sex determining region Y-box 2 (SOX2) (Neuromics # GT 15098)...

Leonard Y.M. Cheung, Hideyuki Okano, Sally A. Camper. Sox21 deletion in mice causes postnatal growth deficiency without physiological disruption of hypothalamic-pituitary endocrine axes. Molecular and Cellular Endocrinology. Available online 8 September 2016.  DOI 10.1016/j.mce.2016.09.005

... The antibodies used in this study were: goat anti-GFP (Abcam Ab5450) at 1:1000; rabbit anti-rat GH (National Hormone and Peptide Program (NHPP), Torrance, CA) at 1:100; rabbit anti-human POMC (NHPP) at 1:150; goat anti-human SOX2 (Neuromics GT15098) at 1:100 ...

Hitoshi Niwa, Akira Nakamura, Makoto Urata, Maki Shirae-Kurabayashi, Shigehiro Kuraku, Steven Russell and Satoshi Ohtsuka. The evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in mouse ES cells. BMC Evolutionary Biology 2016 16:173 DOI: 10.1186/s12862-016-0755-4

Immunostaining
Cells were fixed with 4 % paraformaldehyde in PBS for 30 min at 4°C, followed by permealization with 0.2 % TritonX100 in PBS for 10 min at RT. These cells were incubated with the following primary antibodies overnight at 4°C; mouse monoclonal anti-Sox2 (R&D Systems, MAB2018), 1:1000; goat polyclonal anti-Sox2 (Neuromics, GT15098), 1:1000; mouse monoclonal anti-Oct3/4 (Santa Cruz, 5279), 1:1000; rat monoclonal anti-Nanog (e-Bioscience, MLC-51), 1:1000; rabbit polyclonal anti-Klf4 (Santa Cruz, 20691), 1:300; anti-SoxN antisera (Ferrero et al., 2014), 1:300; rabbit polyclonal anti-Sox1 (Chemicon, ab5768), 1:300; rabbit polyclonal anti-Sox3 (Santa Cruz 20089), 1:300; goat polyclonal anti-Sox15 (Santa Cruz, 17354), 1:300: goat polyclonal anti-Sox17 (R&D Systems, AF1924), 1:300. After washing, cells were incubated with appropriate secondary antibodies conjugated with Alexa-488 for 1 h at RT with Hoechst 33258 and fluorescent images were taken on an Olympus OX-71 equipped with a CCD camera.

Julio Castaño, Cristina Morera, Borja Sesé, Stephanie Boue, Carles Bonet-Costa, Merce Martí, Alicia Roque, Albert Jordan, Maria J. Barrero. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells. Published: February 18, 2016DOI: 10.1371/journal.pone.0149502.

...SOX2 (Neuromics GT15098)...

Naoko Kanno, Masashi Higuchi, Saishu Yoshida, Hideji Yako, Mo Chen, Hiroki Ueharu, Naoto Nishimura, Takako Kato, Yukio Kato. Expression studies of neuronatin in prenatal and postnatal rat pituitary. Cell and Tissue Research pp 1-16 First online: 27 November 2015

... 2011), mouse antiserum against hu- man PRRX2 (1:1000 dilution; Abnova, Taipei City, Taiwan), mouse monoclonal IgG against rat PIT1 (1:50 dilution; Abcam), goat IgG against human SOX2 (1:500 dilution; Neuromics, Edina, Minn., USA), mouse monoclonal IgG against rat ...

Fabio A. Mendes , Juliana M. Coelho Aguiar , Suzana A. Kahn, Alice H. Reis, Luiz Gustavo Dubois, Luciana Ferreira Romão, Lais S. S. Ferreira, Hervé Chneiweiss, Vivaldo Moura Neto, José G. Abreu. Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro. Published: August 4, 2015DOI: 10.1371/journal.pone.0133689.

...monoclonal anti-Sox2 (Neuromics, Acris, Germany) in blocking solution were incubated with the membranes overnight at 4°C, followed by incubation with Peroxidase-conjugated anti-rabbit IgG and peroxidase-conjugated anti-mouse IgG secondary antibodies...

Masashi Higuchi, Takako Kato, Saishu Yoshida, Hiroki Ueharu, Naoto Nishimura, Yukio Kato. PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are involved in vasculogenesis during rat embryonic pituitary development. Cell and Tissue Research March 2015.

...goat antibody against human SOX2 (dilution 1:400; Neuromics, Minn., USA)...

Chung-Wei Lee, Qian Zhan, Cecilia Lezcano, Markus H Frank, John Huang, Allison R Larson, Jennifer Y Lin, Marilyn T Wan, Ping-I Lin, Jie Ma, Sonja Kleffel, Tobias Schatton, Christine G Lian and George F Murphy. Nestin depletion induces melanoma matrix metalloproteinases and invasion. Laboratory Investigation, (3 November 2014) | doi:10.1038/labinvest.2014.130.

SOX2 15 (1:200, Neuromics, Edina, MN, USA...

Xiaoling Wang, Yingjia Wang, He Huang, Buyuan Chen, Xinji Chen, Jianda Hu, Tammy Chang, Ren-Jang Lin, Jiing-Kuan Yee Precise Gene Modification Mediated by TALEN and Single-Stranded Oligodeoxynucleotides in Human Cells. Published: April 01, 2014DOI: 10.1371/journal.pone.0093575.

... Sox2 (1:100, Neuromics, Minneapolis, MN)...

Emma J. Stringer, Isabelle Duluc, Thoueiba Saandi, Irwin Davidson, Monika Bialecka, Toshiro Sato, Nick Barker, Hans Clevers, Catrin A. Pritchard, Doug J. Winton, Nicholas A. Wright, Jean-Noel Freund, Jacqueline Deschamps and Felix Beck. Cdx2 determines the fate of postnatal intestinal endoderm. Published online before print December 21, 2011, doi: 10.1242/dev.070722

...Sox2 (Neuromics; 1:2500)...

S. Yoshida1, T. Kato1, H. Yako, T. Susa, L.-Y. Cai, M. Osuna, K. Inoue and Y. Kato. Significant Quantitative and Qualitative Transition in Pituitary Stem/Progenitor Cells Occurs during the Postnatal Development of the Rat Anterior Pituitary. Journal of Neuroendocrinology. 23, 933–943. copyright 2011 Blackwell Publishing Ltd.

...goat IgG against human SOX2(dilution 1 : 500; Neuromics, Edina, MN, USA)...

O Leis, A Eguiara, E Lopez-Arribillaga, M J Alberdi, S Hernandez-Garcia, K Elorriaga, A Pandiella, R Rezola and A G Martin. Sox2 expression in breast tumours and activation in breast cancer stem cells. O Leis, A Eguiara, E Lopez-Arribillaga, M J Alberdi, S Hernandez-Garcia, K Elorriaga, A Pandiella, R Rezola and A G Martin

...SOX2 (Neuromics GT15098)...

Alvaro C. Laga, Qian Zhan,Carsten Weishaupt, Jie Ma, Markus H. Frank, George F. Murphy. SOX2 and nestin expression in human melanoma: an immunohistochemical and experimental study. Experimental Dermatology. Volume 20, Issue 4, pages 339–345, April 2011. DOI: 10.1111/j.1600-0625.2011.01247.x

... Sections were incubated with SOX2 antibody (Neuromics)...

Marc R. Del Bigio. Cell proliferation in human ganglionic eminence and suppression after prematurity-associated haemorrhage Brain. first published on Apr 7, 2011 as doi: doi:10.1093/brain/awr052

....Goat polyclonal anti-human SOX2; 1/500 dilution...

Alvaro C. Laga, Chiou-Yan Lai, Qian Zhan, Susan J. Huang, Elsa F. Velazquez, Qinghong Yang, Mei-Yu Hsu and George F. Murphy. Expression of The Embryonic Stem Cell Transcription Factor SOX2 in Human Skin-Relevance to Melanocyte and Merkel Cell Biology. American Journal of Pathology. 2010;176:903-913.DOI: 10.2353/ajpath.2010.090495

...For double-label immunofluorescence staining, sections were stained with anti-SOX2 (Neuromics)...

Pia Montanucci, Giuseppe Basta, Riccardo Calafiore. Tissue Engineering Part A-In Vitro–Cultured Human Islet Cell Monolayers: Stemness Markers and Insulin Recovery upon Streptozotocin Exposure -Not available-, ahead of print. doi:10.1089/ten.tea.2009.0124.

 .... mouse anti-Sox-2 (1:100) from Neuromics...

Angel C.Y. Mak, Irene Y.Y. Szeto, Bernd Fritzsch and Kathryn S.E. Cheah. Differential and overlapping expression pattern of SOX2 and SOX9 in inner ear development. doi:10.1016/j.gep.2009.04.003.

...1:600 for goat anti-SOX2 (Neuromics GT15098)...

Saishu Yoshida, Takako Kato, Takao Susa, Li-yi Cai, Michie Nakayama, Yukio Kato. PROP1 coexists with SOX2 and induces PIT1-commitment cells. Biochemical and Biophysical Research Communications, Volume 385, Issue 1, 17 July 2009, Pages 11-15

...SOX2 (1:500 dilution, Neuromics, Edina, MN, USA)...

Daisuke Nonaka. Differential Expression of SOX2 and SOX17 in Testicular Germ Cell Tumors. Am J Clin Pathol, May 2009; 131: 731 - 736.

...tissue samples using SOX2 (rabbit polyclonal, dilution 1:70, citrate buffer; Neuromics, Edina, MN) and SOX17 (goat polyclonal, dilution 1:500, citrate buffer; Neuromics) with the avidin-biotin peroxidase method in a DAKO AutoStainer...

Trond Aasen, Angel Raya, Maria J Barrero, Elena Garreta, Antonella Consiglio1, Federico Gonzalez, Rita Vassena1, Josipa Bili1, Vladimir Pekarik1, Gustavo Tiscornia1, Michael Edel1, Stéphanie Boué & Juan Carlos Izpisúa Belmonte. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nature Biotechnology 26, 1276 - 1284 (2008).

...Sox2 (Neuromics GT15098) ...