antibody against hs  (AMS Biotechnology)

Journal: bioRxiv

Article Title: Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation

doi: 10.1101/2025.08.12.669988

Figure Lengend Snippet: N- deacetyl HS and N- sulfo HS are in part separately clustered on cell membranes in the neural plate. (A) Strategy for double staining of N -deacetyl and N -sulfo HS chains in the neural plate. Stage 14 embryos were stained with anti- N- deacetyl HS (JM-403, mouse IgM) and a Cy3-conjugated monovalent Fab fragment of anti-mouse IgM to avoid cross-reaction. Then, embryos were stained with anti- N- sulfo HS (HepSS-1, mouse IgM) conjugated with Alexa Fluor 488. (B) Localization of N -deacetyl and N -sulfo HS in the neural plate (stage 14). The cyan dotted arrow indicates a cell boundary for the line plot of N -deacetyl and N -sulfo HS intensities in (C). (C) Comparison of localization of N -deacetyl and N -sulfo HS chains. The magenta dotted line indicates the normalized intensity of N- deacetyl HS staining and the green line indicates the normalized intensity of N- sulfo HS staining along the cell boundary shown in (B). Magenta and green arrowheads indicate positions where N -deacetyl and N -sulfo HS are highly accumulated, respectively. Fluorescent intensities were normalized with Min-Max normalization in the plot. (D) Correlation coefficients between localization of N -deacetyl HS and N -sulfo HS in the neural plate. Numbers of embryos (N) and numbers of cell boundaries (n) are as indicated. Scale bar, 10μm.

Article Snippet: Primary antibodies and their dilutions were as follows: anti-Wnt11 (rabbit polyclonal IgG, in-house preparation, 1/2000, or mouse monoclonal IgG2b, in-house preparation, 1/10 ), anti-Vangl2 (HPA027043, Sigma, rabbit polyclonal IgG, 1/200), anti-Fzd7 (ab64636, Abcam, rabbit polyclonal IgG, 1/200), anti-Phospho-Vangl2-T78/S79/S82 (AP1206, ABclonal, rabbit polyclonal IgG, 1/1000), anti-HA (11867423001, Roche, 3F10, rat monoclonal IgG1, Roche, 1/2000), anti-C-Cadherin (6B6, Developmental Studies Hybridoma Bank, mouse monoclonal IgG1, 1/50), anti-ZO1 (33-9100, Invitrogen, ZO1-1A12, mouse monoclonal IgG1, 1/200) anti- N -acetyl HS (in-house preparation, NAH46, mouse monoclonal IgM, 1/50 ), anti- N -sulfo HS (in-house preparation, HepSS-1, mouse monoclonal IgM, 1/400 ), and anti- N -deacetyl HS (370730-1, Amsbio, JM-403, mouse monoclonal IgM, 1/200).

Techniques: Double Staining, Staining, Comparison

Journal: bioRxiv

Article Title: Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation

doi: 10.1101/2025.08.12.669988

Figure Lengend Snippet: Polarized localization of HS chains in the neural plate, depends on PCP formation. (A and B) Spatio-temporal distribution of HS chains in the neural plate at Stages 12 (A) and 14 (B), corresponding to a 2-3-h interval. A relatively anterior region is presented (see also for the observed region). Polarization of HS chains (red rose diagram) was compared with WGA (blue rose diagram). Statistical analysis was performed with the Kuiper 2 sample test. Numbers of embryos (N) and numbers of cells (n) are as indicated. (C) Comparison of HS-chain polarization between Stages 12 and 14. Statistical analysis was performed with the Kuiper 2 sample test. (D) Effect of vangl2 knockdown on HS chains in the neural plate (Stage 14). MOs and vangl2 mRNA with tracer (FITC-dextran; 8.3 ng/embryo) were co-injected into the right dorsal blastomere at the 4-cell stage. Boundaries of tracer-negative and -positive area are indicated with dashed lines. (E) Quantification of membrane localization of HS chains with vangl2 knockdown (D). For statistical analysis, the Wilcoxon rank-sum test was performed. Numbers of embryos (N) and numbers of sub regions (n) are as indicated. (F) Increase of N -deacetyl and N -sulfo HS chains on Vangl2 overexpressing cells (Stage 14). mRNA of vangl2 and membrane tracer (mRuby2-KRas) were co-injected into the right dorsal blastomere of 4-cell embryos. (G) Quantification of membrane localization of HS chains with Vangl2 overexpression (F). For statistical analysis, the Shapiro-Wilk test was initially performed to assess data normality, and t -test was performed after the test of homogeneity of variance by F -test. Numbers of embryos (N) and numbers of cell boundaries (n) are as indicated. Amounts of mRNAs/MOs: vangl2 : 10 pg/embryo (D and E), 100 pg/embryo (F and G); mRuby2-kras : 100 pg/embryo (F and G); vangl2 MOs and std MO: 14 ng/embryo (D and E). Scale bars, 20 μm (A and B), 50 μm (D and F).

Article Snippet: Primary antibodies and their dilutions were as follows: anti-Wnt11 (rabbit polyclonal IgG, in-house preparation, 1/2000, or mouse monoclonal IgG2b, in-house preparation, 1/10 ), anti-Vangl2 (HPA027043, Sigma, rabbit polyclonal IgG, 1/200), anti-Fzd7 (ab64636, Abcam, rabbit polyclonal IgG, 1/200), anti-Phospho-Vangl2-T78/S79/S82 (AP1206, ABclonal, rabbit polyclonal IgG, 1/1000), anti-HA (11867423001, Roche, 3F10, rat monoclonal IgG1, Roche, 1/2000), anti-C-Cadherin (6B6, Developmental Studies Hybridoma Bank, mouse monoclonal IgG1, 1/50), anti-ZO1 (33-9100, Invitrogen, ZO1-1A12, mouse monoclonal IgG1, 1/200) anti- N -acetyl HS (in-house preparation, NAH46, mouse monoclonal IgM, 1/50 ), anti- N -sulfo HS (in-house preparation, HepSS-1, mouse monoclonal IgM, 1/400 ), and anti- N -deacetyl HS (370730-1, Amsbio, JM-403, mouse monoclonal IgM, 1/200).

Techniques: Comparison, Knockdown, Injection, Membrane, Over Expression

Journal: bioRxiv

Article Title: Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation

doi: 10.1101/2025.08.12.669988

Figure Lengend Snippet: Deacetyl HS and N- sulfo HS can be polarized in the ectopically established PCP directed by exogenous Wnt11. (A) Schematic image of injection. mRNAs of GFP-pk3 mixed with ha-vangl2 and wnt11 or tracer (mRuby2-KRas) were injected into adjacent animal-ventral blastomeres of 32-cell stage embryos. (B and C) Localization of GFP-Pk3, Wnt11, and N -deacetyl HS (B) or N -sulfo HS (C) with (lower panels) or without exogenous Wnt11 (upper panels) (Stage 14). Rabbit anti-Wnt11 antibody was generated and evaluated to visualize Wnt11 . Co-localization of GFP-Pk3, Wnt11, and HS chains are indicated with arrowheads. (D and E) Quantification of correlation coefficients between HS chains and Wnt11 (D) or GFP-PK3 (E) in the ectopically established PCP or Wnt11 or core PCP components-only overexpression (see also Figures S6A and S6B). All data were performed the Shapiro-Wilk test to assess data normality. For pairwise comparison, the Wilcoxon rank-sum test was performed for N -sulfo HS in (D), and for N -acetyl and N -deacetyl HS in (E). For N -acetyl and N -deacetyl HS in (D) and N -sulfo HS in (E), a t -test was performed after the test of homogeneity of variance with an F -test. For multiple comparison in (D) and in (E, core PCP components-only overexpression), the Kruskal-Wallis test for pre-analysis and the Steel-Dwass-Critchlow-Fligner test were performed. For multiple comparison in (E, ectopically established PCP), Tukey’s HSD test was performed. For (D), numbers of embryos (N) and numbers of cell boundaries (n) are as indicated. For (E), numbers of embryos (N) and numbers of cells (n) are as indicated. Amounts of mRNAs: GFP-pk3 , 100 pg/embryo (B - E); ha-vangl2 , 50 pg/embryo (B and C); vangl2 , 50 pg/embryo (D and E); wnt11 , 250 pg/embryo (B and C); wnt11-4ha , 250 pg/embryo (D and E); mRuby2-kras , 100 pg/embryo (B - E); mEGFP-kras , 100 pg/embryo (D and E). Scale bars, 50 μm (B and C).

Article Snippet: Primary antibodies and their dilutions were as follows: anti-Wnt11 (rabbit polyclonal IgG, in-house preparation, 1/2000, or mouse monoclonal IgG2b, in-house preparation, 1/10 ), anti-Vangl2 (HPA027043, Sigma, rabbit polyclonal IgG, 1/200), anti-Fzd7 (ab64636, Abcam, rabbit polyclonal IgG, 1/200), anti-Phospho-Vangl2-T78/S79/S82 (AP1206, ABclonal, rabbit polyclonal IgG, 1/1000), anti-HA (11867423001, Roche, 3F10, rat monoclonal IgG1, Roche, 1/2000), anti-C-Cadherin (6B6, Developmental Studies Hybridoma Bank, mouse monoclonal IgG1, 1/50), anti-ZO1 (33-9100, Invitrogen, ZO1-1A12, mouse monoclonal IgG1, 1/200) anti- N -acetyl HS (in-house preparation, NAH46, mouse monoclonal IgM, 1/50 ), anti- N -sulfo HS (in-house preparation, HepSS-1, mouse monoclonal IgM, 1/400 ), and anti- N -deacetyl HS (370730-1, Amsbio, JM-403, mouse monoclonal IgM, 1/200).

Techniques: Injection, Generated, Over Expression, Comparison