mouse monoclonal anti sox2  (R&D Systems)

Journal: Toxicology Reports

Article Title: In utero arsenic exposure increases DNA damage and gene expression changes in umbilical cord mesenchymal stem cells (UC-MSCs) from newborns as well as in UC-MSC differentiated hepatocytes

doi: 10.1016/j.toxrep.2022.09.002

Figure Lengend Snippet: Immunohistochemical detection of MSC markers and DNA damage (8-OHdG and 8-nitroguanine) in paraffin-embedded umbilical cord sections. Paraffin-embedded sections of umbilical cord samples from 100 arsenic-exposed newborns were subjected to immunohistochemical detection of MSC markers and 8-OHdG and 8-nitroguanine. The drawing picture of umbilical cord composition and the staining procedure for MSC markers and 8-OHdG and 8-nitroguanine in umbilical cord cross-sections were modified from a study by Shugar et al. (A) (WJ: Wharton’s jelly). Representative images showing localization of MSC markers in Wharton’s jelly; CD73 (red), CD90 (yellow), and CD105 (orange) in umbilical cord tissue as judged by co-localization of DAPI (blue) as indicated in merge (white) (B-upper). Representative images showing localization of 8-OHdG (green), and 8-nitroguanine (pink) in umbilical cord MSCs as judged by co-localization of DAPI (blue) as indicated in merge (white) (B-lower). Cells were viewed at 200× magnification.

Article Snippet: Secondly, Alexa Fluor®750 goat immunoglobulin G (IgG) anti-rabbit secondary antibody (1:500, Abcam, UK), Alexa Fluor®594 goat IgG anti-mouse secondary antibody (1:500, Molecular Probes, USA), mouse monoclonal anti-human CD73 conjugated to PE (1:50, Abcam), mouse monoclonal anti-human CD105 conjugated to PerCP-CyTM5.5 (1:50, BD PharmigenTM, USA), and mouse monoclonal anti-8-OHdG conjugated to Alexa Fluor®488 (1:100, Santa Cruze Biotechnology, USA) were incubated for 1 h in the dark at 37 °C.

Techniques: Immunohistochemical staining, Staining, Modification

Journal: bioRxiv

Article Title: Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network

doi: 10.1101/2020.03.17.995621

Figure Lengend Snippet: A , B , growth curves of Sox2-deleted cells (green), and of the same cells transduced with the indicated vectors. We performed two independent transductions with Fos alone of Sox2-deleted NSC; the results obtained with the second transduction (not shown) are essentially the same as those depicted here with the first transduction. C , Percentage of dNGFR-positive cells by FACS analysis .

Article Snippet: Coverslips were incubated with the primary anti-SOX2 antibody (mouse monoclonal IgG2a, 1:100, R&D Systems), overnight at 4°C.

Techniques: Transduction

Journal: bioRxiv

Article Title: Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network

doi: 10.1101/2020.03.17.995621

Figure Lengend Snippet: A, Expression levels of selected transcription factor genes among the most downregulated genes in Sox2-deleted NSC (transcripts per million; data from ). B, Scheme of the Sox2 target transduction experiment and of the lentiviral vector

Article Snippet: Coverslips were incubated with the primary anti-SOX2 antibody (mouse monoclonal IgG2a, 1:100, R&D Systems), overnight at 4°C.

Techniques: Expressing, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network

doi: 10.1101/2020.03.17.995621

Figure Lengend Snippet: A , B , Expression of Fos (A) and Socs3 (B) in Sox2-deleted NSC(three different mutants, #1, #2, #3) transduced with Sox2-expressing lentivirus. P<6 and p>6 are the average expression values for passages 2-5 and 6-22, respectively. C , Expression of Socs3 in Sox2-deleted cells transduced with Fos. mut: Sox2-deleted. nt: non transduced. In A,B and C, values reported for non-transduced Sox2-deleted NSC (mut nt) were obtained at the initial passages 1-3, when cells are still actively growing. In A,B,C, expression values are calculated as ratios of Fos or Socs3 mRNA/HPRT mRNA, as determined by qRT-PCR. The values obtained for untransduced mutant NSC (mut nt) are set = 1, and the values obtained for Sox2- or Fos-transduced mutants (mut + Sox2; mut + Fos) are normalized to the corresponding mut nt. The mean of the absolute values obtained for mut nt Fos mRNA/HPRT mRNA are: 25.30 (#1), 16.38 (#2) and 14.63 (#3). The mean of the absolute values obtained for mut nt Socs3 mRNA/HPRT mRNA are: 2.30 (#1), 1.43 (#2) and 1.99 (#3). Histograms in A,B,C represent the mean of results obtained with the following numbers of qRT-PCRs at different passages: mut 1 and 2: 20 qRT-PCRs performed in duplicate for mut nt and mut+Sox2 p>6, and 13 qRT-PCRs performed in duplicate for mut nt and mut+Sox2 p<6; mut 3: 10 qRT-PCRs for nt and mut+Sox2 p>6; 3 qRT-PCRs for mut nt and mut+Sox2 p<6. ***p <0.001; *p<0.05, paired t-test.

Article Snippet: Coverslips were incubated with the primary anti-SOX2 antibody (mouse monoclonal IgG2a, 1:100, R&D Systems), overnight at 4°C.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Mutagenesis

Journal: bioRxiv

Article Title: Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network

doi: 10.1101/2020.03.17.995621

Figure Lengend Snippet: CUT&RUN targeting different transcription factors in neurosphere cells. CUT&RUN was performed on 100,000 neurosphere cells per antibody. Tracks show a duplicate experiment (replicates marked with • or ○), one of which also shows an anti-JUN antibody. Peaks called by SEACR are shown as colored boxes. Location of peaks called using ChIP-seq targeting SOX2 in neurospheres are indicated as grey triangles.

Article Snippet: Coverslips were incubated with the primary anti-SOX2 antibody (mouse monoclonal IgG2a, 1:100, R&D Systems), overnight at 4°C.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network

doi: 10.1101/2020.03.17.995621

Figure Lengend Snippet: A , Regulatory relations between Sox2, Fos, and Socs3, in NSC long-term self-renewal control B , Sox2, Fos and Egr2 functional interactions on the Socs3 promoter. For Fos and Egr2 binding data, see [ , ]. For Sox2 binding, see .

Article Snippet: Coverslips were incubated with the primary anti-SOX2 antibody (mouse monoclonal IgG2a, 1:100, R&D Systems), overnight at 4°C.

Techniques: Control, Functional Assay, Binding Assay

Journal: Scientific Reports

Article Title: CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons

doi: 10.1038/s41598-019-43080-2

Figure Lengend Snippet: Generation of the TH-mOrange reporter iPSC line using CRISPR/Cas9-mediated gene editing. ( A ) Scheme describing the recombination steps during the edition process. Blue arrows represent the primers used for the PCR screening procedure. Black triangles represent LoxP sites surrounding the selection cassette. ( B ) Molecular analysis of the correctly targeted clones to confirm proper P2A-mOrange cassette integration and selection cassette excision in the control iPSC line. Full-length gels are included in Fig. . ( C ) Sanger sequencing confirmed successful excision of the LoxP site-flanked cassette. ( D ) Immunofluorescence analysis of representative colonies of the TH reporter (SP_11) iPSC line staining positive for the pluripotency-associated markers NANOG, OCT4 and SOX2 (green) and TRA-1-81, SSEA3 and SSEA4 (red). Scale bar, 50 µm. ( E ) Normal karyotype of the TH reporter control iPSC line.

Article Snippet: Cells were then blocked in Triton X-100 with 3% donkey serum for 2 h. The following antibodies were used: goat anti-Nanog (R&D Systems, Minneapolis, MI; AF1997; 1:50), mouse IgM anti-Tra-1-81 (Merck-Millipore; MAB4381; 1:200), mouse anti-OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279; 1:30), rat IgM anti-SSEA-3 (Developmental Studies Hybridoma Bank [DSHB] Iowa City, IA; MC-631; 1:10), mouse-SOX2 (R&D Systems; MB2018; 1:50), mouse anti-SSEA-4 (DSHB; MC-813-70; 1:100), mouse anti-NESTIN (Santa Cruz; sc-23927; 1:300action), rabbit anti-LMX1A (Millipore; ab10533; 1:1000), goat anti-EN1 (Santa Cruz; sc-46101; 1:200), mouse anti-TUJ1 (BioLegend, San Diego, CA; 801202; 1:500), chicken anti-MAP2ab (Abcam ab5392; 1:1000) goat anti-FOXA2 (R&D Systems; AF2400; 1:50), rabbit anti-TH (Santa Cruz Biotechnology; sc-14007; 1:500), sheep anti-TH (Pel-Freez Biologicals, Rogers, AR; P60101-0 1:500), rabbit anti-mRFP (Abcam, Cambridge, NA; ab34771; 1:400), rabbit anti-GIRK2 (Sigma-Aldrich; P8122; 1:40).

Techniques: CRISPR, Selection, Clone Assay, Control, Sequencing, Immunofluorescence, Staining