Journal: Frontiers in Neuroscience

Article Title: A Guide to Extract Spinal Cord for Translational Stem Cell Biology Research: Comparative Analysis of Adult Human, Porcine, and Rodent Spinal Cord Stem Cells

doi: 10.3389/fnins.2020.00607

Figure Lengend Snippet: Descriptions of the antibodies used for NSPC characterization including antibody specificity, dilution used, and source.

Article Snippet: β-iii tubulin , Cytoskeletal protein abundant in neural precursors , 1:500 , STEMCELL Technologies.

Techniques: Membrane, Marker

Journal: Frontiers in Neuroscience

Article Title: A Guide to Extract Spinal Cord for Translational Stem Cell Biology Research: Comparative Analysis of Adult Human, Porcine, and Rodent Spinal Cord Stem Cells

doi: 10.3389/fnins.2020.00607

Figure Lengend Snippet: Human, porcine, and rodent primary NSPCs grew in EFH proliferate (BrdU + ) and express neural stem cell marker Sox2. Upon differentiation, NSPCs are multipotent and generate β-iii tubulin + neural precursors, GFAP + astrocytes, and O4 + oligodendrocytes. No O4 + staining was observed from porcine NSPCs. Scale bar = 100 μm.

Article Snippet: β-iii tubulin , Cytoskeletal protein abundant in neural precursors , 1:500 , STEMCELL Technologies.

Techniques: Marker, Staining

Journal: Molecular Oncology

Article Title: Downregulation of miR‐326 and its host gene β‐arrestin1 induces pro‐survival activity of E2F1 and promotes medulloblastoma growth

doi: 10.1002/1878-0261.12800

Figure Lengend Snippet: Biological effects of ectopic expression of miR‐326 and ARRB1 in MB CSCs. (A–C) MB CSCs were assayed 48 h after transfection with miR‐326 and ARRB1 , individually or combined. Mock‐transfected cells served as controls. Ectopic expression of ARRB1 and/or miR‐326 in these cells (A) reduced proliferation (MTT assay) (24 h: vs. CTRL: miR‐326+ P = 0.0072; ARRB1‐HA+ P = 0.0245; miR‐326+ARRB1‐HA+ P = 0.0015; 48 h: vs. CTRL: miR‐326+ P = 0.001; ARRB1‐HA+ P = 0.0005; miR‐326+ARRB1‐HA+ P < 0.0001), (B) diminished the frequency of oncosphere‐forming cells (vs. miR‐326‐ARRB1‐HA−: miR‐326+ P = 0.001; ARRB1‐HA+ P = 0.0005; miR‐326+ARRB1‐HA+ P = 0.0003), (C) decreased NANOG expression and increased the expression of PARP‐C (miR‐326 levels: miR‐326+ P < 0.0001; miR‐326+ARRB1‐HA+ P = 0.0002 vs. miR‐326‐ARRB1‐HA−). (D) qRT‐PCR revealed significantly increased expression of neuronal and glial differentiation markers ( βIII tubulin and GFAP , respectively) only in MB CSCs overexpressing miR‐326 (alone or with ARRB1 ) (vs. Mock βIII tubulin: miR‐326 P = 0.0027; miR‐326 and ARRB1 P = 0.046, GFAP: miR‐326 P = 0.0002; miR‐326 and ARRB1 P = 0.011). Data represent means ± SD from five independent experiments. Statistics: One‐way ANOVA and two‐way ANOVA, * P < 0.05; **P < 0.01; *** P < 0.001; **** P < 0.0001 vs. indicated controls.

Article Snippet: Cell was stained with mouse monoclonal antibodies against βIII tubulin (MAB 1637; Merck, Darmstadt, Germany) and GFAP (MAB360).

Techniques: Expressing, Transfection, MTT Assay, Quantitative RT-PCR

Journal: Molecular Vision

Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

doi:

Figure Lengend Snippet: Transduction of rAAV-hSyn-GFP and rAAV-hSyn-Brn3b and overexpression of Brn3b in RGCs of Brown Norway rats. A : Immunohistochemical analyses for green fluorescent protein (GFP; green) in frozen retinal sections from rats injected with the recombinant adenoassociated virus–hSyn–green fluorescent protein (rAAV-hSyn-GFP) virus. Binding of the GFP antibody was detected using an Alexa 488-conjugated donkey anti-rabbit immunoglobulin G (IgG) antibody. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to detect nuclei. B : Brn3b (pseudogreen) and βIII-tubulin (pseudored) immunostaining in retinal frozen sections from rats intravitreally injected with either rAAV-hSyn-GFP or rAAV-hSyn-Brn3b. The immunostaining was detected by using corresponding Alexa 546 (pseudogreen) and Alexa 647 (pseudored)-conjugated donkey anti-immunoglobulin G (IgG) secondary antibodies. Cells were counterstained with DAPI (blue) to detect cell nuclei. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment. Scale bar indicates 20 µm. C : Representative images of the GCL showing Brn3b staining in rats injected with rAAV-hSyn-GFP or rAAV-hSyn-Brn3b. D : Plot of the ratio of fluorescence intensity of Brn3b immunostaining in RGCs between left (L: intravitreally injected) and right (R: contralateral) eyes in 24 regions. The L/R ratio was compared between rats injected with rAAV-hSyn-GFP and rAAV-hSyn-Brn3b. A significant increase in Brn3b staining in RGCs was observed in rats that overexpressed Brn3b compared to the control vector. Fluorescent intensity values are shown as mean ± standard error of the mean (SEM), n = 6. The Mann–Whitney rank-sum test was used for statistical analysis (*p<0.002).

Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

Techniques: Transduction, Over Expression, Immunohistochemical staining, Injection, Recombinant, Virus, Binding Assay, Immunostaining, Staining, Fluorescence, Control, Plasmid Preparation, MANN-WHITNEY

Journal: Molecular Vision

Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

doi:

Figure Lengend Snippet: Transcription factor Brn3b-mediated changes in Bcl-2 and Bcl-xL expression in RGCs of Brown Norway rats. A : Bcl-2 (pseudogreen), and βIII-tubulin (pseudored) expression in retinal sections from Brown Norway rat eyes injected with either the recombinant adenoassociated virus–hSyn–green fluorescent protein (rAAV-hSyn-GFP) virus (control) or the rAAV-hSyn-Brn3b virus, detected with secondary antibodies conjugated with Alexa 546 (pseudogreen) or Alexa 647 (pseudored) dye. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue) to detect cell nuclei. B : Magnified image of the retinal ganglion cell (RGC) layers showing Bcl-2 immunostaining of RGCs of retinas transduced with either rAAV-hSyn-GFP or rAAV-hSyn-Brn3b. Scale bar indicates 20 µm. C : Ratio of fluorescence intensity for Bcl-2 staining was measured at 24 different regions in the ganglion cell layers using ImageJ. The fluorescence intensity ratios are shown as mean ± standard error of the mean (SEM), n = 6. A statistically significant increase in the intensity of Bcl-2 staining was found in the RGCs of the rats injected with rAAV-hSyn-Brn3b, compared to those injected with rAAV-hsyn-GFP. Statistical analysis was performed with the Mann–Whitney rank-sum test (*p<0.002). D : Bcl-xL (pseudogreen) and βIII-tubulin (pseudored) immunostaining in frozen retinal sections from rats intravitreally injected with either rAAV-hSyn-GFP or rAAV-hSyn-Brn3b.

Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

Techniques: Expressing, Injection, Recombinant, Virus, Control, Immunostaining, Transduction, Fluorescence, Staining, MANN-WHITNEY

Journal: Molecular Vision

Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

doi:

Figure Lengend Snippet: Transcription factor Brn3b promoted an increase in the levels of p-AKT in retinas of rats injected with rAAV-hSyn-Brn3b. A : Immunostaining for p-AKT (pseudogreen), βIII-tubulin (pseudored) expression in retinal sections from Brown Norway rats intravitreally injected with either the recombinant adenoassociated virus–hSyn–green fluorescent protein (rAAV-hSyn-GFP; vector control) or rAAV-hSyn-Brn3b virus. The immunostaining was detected using corresponding Alexa 546 (pseudogreen) or Alexa 647 (pseudored) conjugated secondary antibody. Scale bar indicates 20 µm. B : A magnified view of the retinal ganglion cell (RGC) layers of retinas transduced with either rAAV-hSyn-GFP or rAAV-hSyn-Brn3b. C : A significant 2.4-fold increase in p-AKT expression was observed in the RGCs of rats injected with rAAV-hSyn-Brn3b. Ratios of fluorescence intensity values are shown in mean ± standard error of the mean (SEM), n = 6. The Mann–Whitney rank-sum test was used for statistical analysis (*p<0.002).

Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

Techniques: Injection, Immunostaining, Expressing, Recombinant, Virus, Plasmid Preparation, Control, Transduction, Fluorescence, MANN-WHITNEY

Journal: Molecular Vision

Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

doi:

Figure Lengend Snippet: AAV-mediated overexpression of Brn3b in RGCs of Brown Norway rats with elevated IOP following intravitreal injection of rAAV-CMV-Brn3b. A : Representative images show Brn3b (pseudogreen) and βIII-tubulin (pseudored) immunostaining in retinas of rats intravitreally injected with either recombinant adenoassociated virus–cytomegalovirus–green fluorescent protein (rAAV-CMV-GFP) or rAAV-CMV-Brn3b. Brn3b staining was detected mainly in the GCL. B : Intraocular pressure (IOP) elevation profile in Brown Norway rats administered rAAV-CMV-GFP. C : IOP elevation profile in Brown Norway rats administered rAAV-CMV-Brn3b. IOP was elevated in one eye (closed circles), while the other eye served as the contralateral control eye (open circles). IOP elevation was performed in three Brown Norway rats followed by intravitreal injection with either rAAV-CMV-GFP or rAAV-CMV-Brn3b. IOP values were plotted as mean ± standard error of the mean (SEM; solid line, IOP-elevated and virus-injected eye; dashed line, untreated contralateral eye). Asterisk indicates p<0.001 statistically significant elevation of IOP in the IOP-elevated eye compared with contralateral eye using the Student t test. Scale bar indicates 20 µm.

Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

Techniques: Over Expression, Injection, Immunostaining, Recombinant, Virus, Staining, Control

Journal: Molecular Vision

Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

doi:

Figure Lengend Snippet: rAAV-2-mediated overexpression of Brn3b produced an increase in Bcl-2 expression in the RGCs of Brown Norway rats with elevated IOP. A : Brown Norway rats with elevated intraocular pressure (IOP) were intravitreally injected with either recombinant adenoassociated virus–cytomegalovirus promoter–green fluorescent protein (rAAV-CMV-GFP) or rAAV-CMV-Brn3b. Retinal sections obtained were immunostained for Bcl-2 (pseudogreen) and βIII-tubulin (pseudored). B : The L/R ratio of fluorescence intensity of Bcl-2 immunostaining in retinal ganglion cells (RGCs) from rats intravitreally injected with either rAAV-CMV-GFP or rAAV-CMV-Brn3b is plotted. Densitometry analysis shows a significant increase in Bcl-2 expression in RGCs transduced with rAAV-CMV-Brn3b, compared to RGCs transduced with rAAV-CMV-GFP in the rats with elevated IOP. Student t test was used for statistical analysis (*p<0.05). Values are represented as mean ± standard error of the mean (SEM), n = 3. Scale bar indicates 20 µm. C : Brown Norway rats had elevated IOP and subsequently were intravitreally injected with either rAAV-CMV-GFP or rAAV-CMV-Brn3b. Retinal sections obtained were immunostained for Bcl-xL (pseudogreen) and βIII-tubulin (pseudored). D : A plot of the L/R ratio of fluorescence intensities of Bcl-xL immunostaining in the RGCs of rats intravitreally injected with either rAAV-CMV-GFP or rAAV-CMV-Brn3b. An increasing trend in Bcl-xL expression (not statistically significant) was observed in rats intravitreally injected with rAAV-CMV-Brn3b, compared to those administered the control vector.

Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

Techniques: Over Expression, Produced, Expressing, Injection, Recombinant, Virus, Fluorescence, Immunostaining, Transduction, Control, Plasmid Preparation

Journal: Molecular Vision

Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

doi:

Figure Lengend Snippet: Levels of p-AKT in the retinas of rats with elevated IOP overexpressing Brn3b. A : Immunostaining for p-AKT in retinal ganglion cells (RGCs) of Brown Norway rats intravitreally injected with either the recombinant adenoassociated virus–cytomegalovirus–green fluorescent protein (rAAV-CMV-GFP) or rAAV-CMV-Brn3b following intraocular pressure (IOP) elevation. Retinal sections obtained were immunostained for p-AKT (pseudogreen) and βIII-tubulin (pseudored). B : An increase in immunostaining (not statistically significant) for p-AKT was observed in RGCs overexpressing Brn3b (rAAV-CMV-Brn3b) compared to RGCs overexpressing the control vector (rAAV-CMV-GFP; determined with the L/R ratios of fluorescence intensities of p-AKT staining in RGCs). Values are represented as mean ± standard error of the mean (SEM), n = 3. Scale bar indicates 20 µm.

Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

Techniques: Immunostaining, Injection, Recombinant, Virus, Control, Plasmid Preparation, Fluorescence, Staining

Journal: Tissue Engineering. Part A

Article Title: Stem/Progenitor Cell-Mediated De Novo Regeneration of Dental Pulp with Newly Deposited Continuous Layer of Dentin in an In Vivo Model

doi: 10.1089/ten.tea.2009.0518

Figure Lengend Snippet: Multiple lineage differentiation properties of SCAP and DPSCs compared with BMMSCs. (A, D, G) Odonto/osteogenic induction and Alizarin Red staining of matrix mineralization. Insets: uninduced control. All scale bars including insets: 200 μm. (B, E, H) Adipogenic induction and Oil Red O staining of the accumulated lipid droplets in cells. Inset images: higher magnification views of the adipocyte-like cells from the same culture shown in the main images. Scale bars: main images, 100 μm; insets, 50 μm. (C, F, I) Neurogenic induction. βIII-tubulin: red fluorescence; 4',6-diamidino-2-phenylindole dihydrochloride (DAPI): blue fluorescence. Scale bars: (C) 20 μm; (F, I) 30 μm. (J) Myogenic induction and RT-polymerase chain reaction analysis of gene expression. M, RNA from cultured human myoblasts (obtained from Dr. Wesley M. Jackson, NIH/NIAMS, Bethesda, MD); C, uninduced control; m, induction in myogenic medium. SCAP, stem cells from apical papilla; DPSC, dental pulp stem cells; BMMSCs, bone marrow mesenchymal stem cells.

Article Snippet: After fixation in 100% ice-cold methanol, cells were incubated in blocking buffer (32.5 mM NaCl, 3.3 mM Na 2 HPO 4 , 0.76 mM KH 2 PO 4 , 1.9 mM NaN 3 , 0.1% [w/v] bovine serum albumin, 0.2% [v/v] Triton-X 100, 0.05% [v/v] Tween 20, and 5% goat serum) for 30 min followed by the addition of monoclonal mouse anti-human βIII-tubulin antibodies (Promega, Madison, WI) for 1 h at room temperature.

Techniques: Staining, Control, Fluorescence, Polymerase Chain Reaction, Gene Expression, Cell Culture

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion

doi: 10.1007/s13770-021-00330-7

Figure Lengend Snippet: Antibodies tested for flow cytometry analysis on undifferentiated and differentiated CMC. PE: Phycoerythrin; PE-Cy7: Phycoerythrin-cyanine7

Article Snippet: mouse anti-human βIII Tubulin (TBB3) , Merck Millipore.

Techniques: Flow Cytometry, Control

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion

doi: 10.1007/s13770-021-00330-7

Figure Lengend Snippet: Primer pairs for RT-PCR study

Article Snippet: mouse anti-human βIII Tubulin (TBB3) , Merck Millipore.

Techniques: Sequencing

Journal: Tissue Engineering and Regenerative Medicine

Article Title: Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion

doi: 10.1007/s13770-021-00330-7

Figure Lengend Snippet: A Flow cytometrical analysis of TBB3, MAP2, TH on induced (T14) CMCs (coloured profile) compared to cells cultured in αMEM, NBM, and NBM supplemented with neurogenic factors for 14 days (black profile for all). Indirect staining with FITC-conjugated secondary antibodies was used. For each marker, data were expressed as % positives ± SD of T14 versus C14. B Western Blot analysis on undifferentiated (Undiff), C7, T7 and T14 CMCs using 10 g protein extract and chemiluminescence detection. C Densitometric quantification of Western Blot bands. (* p ≤ 0.05 vs undifferentiated sample). D Immunofluorescence of neuronal lineage markers on induced CMCs (T14) compared to undifferentiated samples (Undiff). Cells were indirectly labeled using FITC-conjugated secondary antibodies and images were acquired with the Leica TCS SP5 confocal microscope. Scale bar: 50 µm

Article Snippet: mouse anti-human βIII Tubulin (TBB3) , Merck Millipore.

Techniques: Cell Culture, Staining, Marker, Western Blot, Immunofluorescence, Labeling, Microscopy