Journal: PLoS ONE

Article Title: Early Acquisition of Neural Crest Competence During hESCs Neuralization

doi: 10.1371/journal.pone.0013890

Figure Lengend Snippet: Antibody purchasing and dilution information.

Article Snippet: p75 , Alomone Labs , Rabbit , 1∶200 IF/1∶300 facs.

Techniques: In Ovo

Journal: PLoS ONE

Article Title: Early Acquisition of Neural Crest Competence During hESCs Neuralization

doi: 10.1371/journal.pone.0013890

Figure Lengend Snippet: Immunostaining of adherent neurospheres and emigrating NCSCs after 3 days on fibronectin-coated plates. ( A ) DAPI staining for nuclei. Note the rosette structures within the adherent neurospheres. ( B ) Sox10 staining is observed in emigrating cells, but not in the adherent neurospheres. ( C ) p75 staining is observed in adherent neurospheres and emigrating cells. ( D ) High magnification of the overlay in B–C. ( E ) Confocal image of emigrating NCSCs confirms the membrane localization of p75. ( F ) HNK-1 staining is observed in adherent neurospheres and emigrating cells. ( G, H ) Adherent neurospheres were manually removed and replated on fibronectin and the second wave of emigrating cells was observed. ( G ) Secondary emigrating cells acquire Sox10, while the majority of cells in the replated neurosphere are Sox10-negative. ( H ) The expression of in the second wave of emigrating cells. Scale bars in A, B, C and D represent 100 µm and 10 µm in E Bars in F,G and H depict 150 µm.

Article Snippet: p75 , Alomone Labs , Rabbit , 1∶200 IF/1∶300 facs.

Techniques: Immunostaining, Staining, Expressing

Journal: PLoS ONE

Article Title: Early Acquisition of Neural Crest Competence During hESCs Neuralization

doi: 10.1371/journal.pone.0013890

Figure Lengend Snippet: ( A ) Day 8 cultures (containing emNCSC and neuroepithelial clusters) can be FACS-sorted based on p75 expression defining three populations: negative, medium and highly positive for p75. The grey profile corresponds to control situation with the secondary antibody only. ( B ) All fractions of cells were positive for Sox10 (85, 70 and 60% respectively for high, medium and negative). ( C ) All fractions can differentiate into neuronal, glial and smooth muscle lineages (p75-negative fraction is shown as an example). p75-negative sorted cells are reactive for; I . GFAP, II . β-3-tubulin, III . nestin, IV . smooth muscle actin. Cell nuclei are visualized by DAPI. Magnifications are 100x. Scale bars 30 µM.

Article Snippet: p75 , Alomone Labs , Rabbit , 1∶200 IF/1∶300 facs.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Early Acquisition of Neural Crest Competence During hESCs Neuralization

doi: 10.1371/journal.pone.0013890

Figure Lengend Snippet: The effect of Noggin (BMP inhibitor) and DKK (Wnt inhibitor) on hESC-derived emNCSCs. Untreated (control) neurosphere cultures are strongly reactive for ( A ) SOX10 (green), ( B ) HNK-1 (green), and ( C ) p75 (red) and negative for the CNS marker ( D ) Olig2 (red). The addition of Noggin to NS medium ablates ( E ) SOX10 (green), ( F ) HNK-1 (green), and ( G ) p75 (red) and induces expression of the CNS marker ( H ) Olig2 (red). Culturing neurospheres from day 0 in media that was collected from 293T cells transfected with a DKK plasmid ablates expression of the NC markers ( I ) SOX10 and ( J ) HNK-1 while the low affinity neutrophin receptor ( K ) p75 was still expressed. The insets in upper left corners show the cell nuclei (DAPI). Scale bars represent 50 µm in A, and C–H, 10 µm in B, and 100 µm in I–K.

Article Snippet: p75 , Alomone Labs , Rabbit , 1∶200 IF/1∶300 facs.

Techniques: Derivative Assay, Marker, Expressing, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Early Acquisition of Neural Crest Competence During hESCs Neuralization

doi: 10.1371/journal.pone.0013890

Figure Lengend Snippet: hESC clusters are suspended for 5 days in NS medium. On day 5 of suspension culture neurospheres were allowed to adhere on fibronectin-coated plates and cultured in NS medium for 3 more days. Adherent neurospheres composed of neuroepithelial rosettes are negative for Sox10 and CD73. Migrating cells are positive for the NC markers Sox10 and CD73 (13%). Both cell types are positive for p75 and HNK-1. Rosettes can be manually excised and replated (dotted arrow) to yield additional waves of Sox10-positive emNCSCs. The adherent rosettes are therefore competent to become NC after they have undergone the EMT. Migratory emNCSCs can be allowed to spontaneously acquire NC lineage fates or can be directed to enrich for certain cell fates by the addition of growth factors.

Article Snippet: p75 , Alomone Labs , Rabbit , 1∶200 IF/1∶300 facs.

Techniques: Cell Culture

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: Expression of NGF and p75NTR fibroblasts derived from control individuals and Rett patients. ( A ) Total RNA was extracted from healthy control (HC-1) and Rett syndrome (RTT-1) fibroblasts, and the expression levels of ngf were measured by qRT-PCR. n = 3 biological replicates. Data represent means ± SD. ( B ) Immunofluorescence and respective quantitative analysis of NGF in HC and RTT. Cells were fixed in 4% PFA and stained with antibodies against NGF (red). DAPI (blue) was employed for nuclear counterstaining. n = 6 biological replicates. ( C ) qRT-PCR analysis of ngfr ( p75NTR ) in HC-1 and RTT-1 fibroblasts. n = 3 biological replicates. ( D ) Immunofluorescence and respective quantitative analysis of p75NTR immunoreactivity in HC-1 and RTT-1 cells. Cells were fixed in 4% PFA and stained with anti-p75NTR (red). DAPI (blue) was used to counterstain nuclei. n = 5 biological replicates. Data are expressed as mean ± SD. Statistical analysis was performed by using the Student’s unpaired t -test. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. DMSO. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm.

Article Snippet: The anti-NGF and anti-p75NTR antibodies were validated using NGF (BLP-N240, Alomone labs, Jerusalem, Israel) and p75NTR (BLP-NT011 Alomone labs, Jerusalem, Israel) blocking peptides.

Techniques: Expressing, Derivative Assay, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Software

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: Effects of p75NTR modulation on the antioxidant response in RTT fibroblasts. Representative Western blot and densitometric analysis of ( A ) SOD1, ( B ) SOD2, ( C ) Catalase, ( D ) TrxR1, and ( E ) Gpx1 in healthy control fibroblasts (HC-1), Rett syndrome fibroblasts (RTT-1) and RTT fibroblasts treated with 0.1 μM of LM11A-31(RTT-1+LM) for 24 h. n = 3–5 biological replicates. GAPDH was used as a loading control. ( F ) Immunofluorescence and quantification of GSH immunoreactivity in HC-1, RTT-1, and RTT-1+LM experimental groups. Cells were fixed in 4% PFA and stained with antibodies against GSH (red). Nuclear staining was performed with DAPI (blue). n = 12 biological replicates. Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test. Statistical significance is indicated as follows: ** p < 0.01; *** p < 0.001. “a” indicates statistical significance vs. HC-1; “b” indicates statistical significance vs. RTT-1. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm.

Article Snippet: The anti-NGF and anti-p75NTR antibodies were validated using NGF (BLP-N240, Alomone labs, Jerusalem, Israel) and p75NTR (BLP-NT011 Alomone labs, Jerusalem, Israel) blocking peptides.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Microscopy, Software

Journal: Biomedicines

Article Title: p75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome

doi: 10.3390/biomedicines12112624

Figure Lengend Snippet: p75NTR modulation reduces IL-6 and IL-8 expression in RTT fibroblasts. Representative immunofluorescence and respective quantitative analysis of ( A ) IL-6 immunoreactivity in control fibroblasts (HC-1), Rett syndrome fibroblasts (RTT-1), and RTT fibroblasts treated with LM11A-31(RTT-1+LM) at the dose of 0.1 μM for 24 h. Cells were fixed in 4% PFA and stained with antibodies against IL-6 (red). DAPI was used to counterstain nuclei. n = 6 biological replicates. Images were acquired using the Leica TCS SP8 confocal microscope and Leica Application Suite X (LAS X) software at 40× magnification. Scale bar: 50 µm. ( B ) ELISA on IL-6 in culture medium from HC-1, RTT-1, and RTT-1+LM groups treated as abovementioned. n = 4 biological replicates. ( C ) IL-8 immunoreactivity (red) and respective quantitative analysis performed on HC-1, RTT-1, and RTT-1+LM fibroblasts treated as in ( A ). DAPI was used for nuclear staining. n = 6 biological replicates. Scale bar: 50 µm. ( D ) ELISA on secreted IL-8 in conditioned medium from HC-1, RTT-1, and RTT-1+LM cells treated as abovementioned. n = 4 biological replicates. Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s post hoc test. Statistical significance is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001. “a” indicates statistical significance vs. HC-1; “b” indicates statistical significance vs. RTT-1.

Article Snippet: The anti-NGF and anti-p75NTR antibodies were validated using NGF (BLP-N240, Alomone labs, Jerusalem, Israel) and p75NTR (BLP-NT011 Alomone labs, Jerusalem, Israel) blocking peptides.

Techniques: Expressing, Immunofluorescence, Control, Staining, Microscopy, Software, Enzyme-linked Immunosorbent Assay