Chenglin Sang,Xuecheng Cao, Fangjing Chen, Xuekang Yang, and Yongxian Zhang. Differential Characterization of Two Kinds of Stem Cells Isolated from Rabbit Nucleus Pulposus and Annulus Fibrosus. Stem Cells International. Volume 2016 (2016), Article ID 8283257, 14 pages. http://dx.doi.org/10.1155/2016/8283257 ...Stem Cell Marker Analysis by

Immunocytochemical Staining

The characterizations of disc stem cells (AFSCs and NPSCs) and disc fibroblasts (DFCs) were examined by immunocytochemical staining. The AFSCs and NPSCs and DFCs at passage 1 were seeded in 12-well plate at a density of 3 × 104 cells/well and cultured for 5 days. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature. The fixed cells were directly reacted with the following antibodies with different dilution for 2 hours at room temperature, such as mouse anti-stage-specific embryonic antigen-4 (SSEA-4; 1 : 500, Cat. # 414000, Invitrogen, Carlsbad, CA), mouse anti-Stro-1 antibody (1 : 350, Invitrogen, Cat. # 398401, Carlsbad, CA), mouse anti-collagen type I (1 : 350, Abcam, Cat. # ab90395, Cambridge, MA), mouse anti-collagen type II (1 : 350, Abcam, Cat. # ab3092, Cambridge, MA), and mouse anti-collagen type III (1 : 350, Abcam, Cat. # ab7778, Cambridge, MA). For octamer-binding transcription factor-4 (Oct-4) and nucleostemin (NS) staining, the fixed cells were further treated with 0.1% triton X-100 for 30 min at room temperature and then washed once with PBS. The triton X-100 treated cells were incubated either with mouse anti-Oct-4 antibody (1 : 350, Cat. #MAB4401, Millipore, Temecula, CA) or with goat anti-nucleostemin antibody (1 : 500, Cat. # GT15050, Neuromics, Edina, MN) for 2 hours at room temperature. After washing the cells three times with PBS, cyanine 3- (Cy3-) conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibody (1 : 500, Cat. # A10521, Invitrogen, Carlsbad, CA) was used for Oct-4, SSEA-4, Stro-1, collagen type I, collagen II, and collagen III determination. In addition, nucleostemin was examined by Cy3 conjugated donkey anti-goat IgG antibody (1 : 500, Cat. # AP180C, Millipore, Temecula, CA). Finally, Hoechst 33342 (1 μg/mL, Cat. # 33270, Sigma, St Louis) was used for total cell counterstaining in each well....

Xiaokui Lv, Qianping Guo, Fengxuan Han, Chunyang Chen, Christopher Ling, Weiguo Chen and Bin Li. Electrospun Poly(l-lactide)/Poly(ethylene glycol) Scaffolds Seeded with Human Amniotic Mesenchymal Stem Cells for Urethral Epithelium Repair. Int. J. Mol. Sci. 2016, 17(8), 1262; doi:10.3390/ijms17081262

....The cell phenotype was characterized by differentiation and immunofluorescence measurements. hAMSCs at passage 0-3 were used for immunofluorescence. Cells were washed three times with PBS and fixed in cold 4% paraformaldehyde for 15 min. Following that, the cells were washed twice with PBS and treated with methanol at -20 °C for 5 min. For Oct-4 immunofluorescence, the fixed cells were washed with PBS three times and blocked with 4% BSA for 30 min before being incubated with mouse anti-human Oct-4 antibody (Cat. # MAB4401, Millipore, Billerica, MA, USA; 1:500 dilution in PBS) overnight at 4 °C. After being washed with PBS, the cells were incubated with Cy3-conjugated goat anti-mouse secondary antibody (A10521, Invitrogen, Carlsbad, CA, USA; 1:1000) for 1 h at room temperature. DNA was visualized by DAPI staining and cells were viewed under a fluorescence inverted microscope (EVOS f1, AMG, Carlsbad, CA, USA USA). For nucleostemin staining, goat anti-human nucleostemin antibody (GT15050, Neuromics, Edina, MN, USA; 1:250) along with Cy3-conjugated donkey anti-goat IgG secondary antibody (AP180C, Millipore; 1:1000) were used....

Lei Zhang, MD, PhD, Shuo Chen, MD, PhD, Peng Chang, MD, PhD, Nirong Bao, MD, Chao Yang, MD, Yufan Ti, MD, Liwu Zhou, MD, PhD and Jianning Zhao, MD, PhD. Harmful Effects of Leukocyte-Rich Platelet-Rich Plasma on Rabbit Tendon Stem Cells In Vitro. Am J Sports Med May 16, 2016. doi: 10.1177/0363546516644718

... and washed with PBS 3 times. The treated cells were reacted with either goat anti-NS antibody (1:250; Cat. No. GT15050; Neuromics Inc) or mouse anti-Oct-4 antibody (1:250; Cat. No. MAB4401; Millipore) overnight at 4°C. For ...

He Huang, Shukui Wang, Jianchao Gui, Haiqi Shen. A study to identify and characterize the stem/progenitor cell in rabbit meniscus.Cytotechnology: Incorporating Methods in Cell Science International Journal of Cell Culture and Biotechnology. ISSN: 0920-9069 (Print) 1573-0778 (Online)

... After washing the cells with PBS, either rabbit anti-Nanog (1:350, Santa Cruz Biotechnology,Inc., cat. # sc-33759, Santa Cruz, CA, USA) or goat anti-nucleostemin (1:400, Neuromics, Cat. #GT15050, Edina, MN, USA) was applied for 2 h at room for 2 h at room temperature. ...

Zhe Ding and He Huang. Mesenchymal stem cells in rabbit meniscus and bone marrow exhibit a similar feature but a heterogeneous multi-differentiation potential: superiority of meniscus as a cell source for meniscus repair. BMC Musculoskeletal Disorders (2015) 16:65 DOI 10.1186/s12891-015-0511-8.

...After removing the medium, the cells were washed with PBS once. MMSCs and BMSCs were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and treated with 0.1% Triton X-100 for 30 min for Nanog and nucleostemin staining. After washing the cells with PBS, either mouse anti-Nanog (1:350, Santa Cruz Biotechnology, Inc., cat. # SC-33759, Santa Cruz, CA) or goat antinucleostemin (1:400, Neuromics, Cat. # GT15050, Edina, MN)...

Zhang J, Wang JH-C (2013) The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study. PLoS ONE 8(8): e71740. doi:10.1371/journal.pone.0071740

... goat anti-mouse nucleostemin (1:350; Neuromics, Edina, MN; Cat. #GT15050)...

Haruhiko Shugo, Takako Ooshio, Masako Naito, Kazuhito Naka, Takayuki Hoshii, Yuko Tadokoro, Teruyuki Muraguchi, Akira Tamase, Noriyuki Uema, Taro Yamashita, Yasunari Nakamoto, Toshio Suda, Shuichi Kaneko, and Atsushi Hirao.Nucleostemin in Injury-Induced Liver Regeneration. Stem Cells and Development. -Not available-, ahead of print. doi:10.1089/scd.2011.0725.

...Membranes were then incubated with a goat anti-NS antibody (1:1,000; Neuromics, Edina, MN)...

Jianying Zhang, Tiffany Pan, Hee-Jeong Im, Freddie H Fu and James HC Wang. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Medicine 2011, 9:68doi:10.1186/1741-7015-9-68.

...goat anti-human nucleostemin (1:350, Cat. # GT15050, Neuromics, Edina, MN)...

Jianying Zhang and James H-C. Wang. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskeletal Disorders. 2010, 11:10doi:10.1186/1471-2474-11-10.

...The staining protocol used goat anti-human nucleostemin antibody (1:300; Neuromics, Cat. No. GT15050) and Cy3-conjugated donkey anti-goat IgG secondary antibody.

Tetsuro Tamaki, Yoshinori Okada, Yoshiyasu Uchiyama, Kayoko Tono, Maki Masuda, Masahiro Nitta, Akio Hoshi, Akira Akatsuka. Skeletal Muscle–Derived CD34+/45- and CD34-/45- Stem Cells Are Situated Hierarchically Upstream of Pax7+. Cells. Stem Cells and Development. August 1, 2008, 17(4): 653-668. doi:10.1089/scd.2008.0070.

...anti-nucleostemin (1:200; overnight; Neuromics, Edina, USA) for proliferative capacity...

...Nucleostemin wasvisualized by a commercial goat polyclonal antibody(Neuromics, Bloomington, MN)...
 

Kevin Chen, Rebecca A. Henry, Stephanie M. Hughes, and Bronwen Connor. Creating a neurogenic environment: The role of BDNF and FGF2 . doi:10.1016/j.mcn.2007.06.004  

...goat anti-nucleostemin (1:10000; Neuromics, MN, USA). ...