anti ps6  (Cell Signaling Technology Inc)

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Representative hematoxylin and eosin (HE) staining of 20μm-thick frozen brain sections showing dysmorphic neurons (DN, empty arrowheads) and balloon cells (BC, filled arrowheads) across FCDII patients (n = 1 section/patient). Scale bar: 100μm. Insets for patients pt6, pt7 and pt10 show a representative BC from distinct cortical regions. b , Immunohistochemical detection of mTOR pathway activation using phosphorylated S6 (pS6-Ser240/244) antibody on 4μm-thick formalin-fixed paraffin-embedded (FFPE) brain sections (n = 1 section/patient). DN (empty arrowheads) and BC (filled arrowheads) exhibit strong pS6 immunoreactivity. Scale bar: 50μm. Insets for patients pt7-9 show representative DN from distinct cortical regions. c , Quantification of grey and white matter proportions in 20μm-thick HE-stained frozen brain sections adjacent to tissue used for single-nucleus RNA sequencing (n = 1 section/patient).

Article Snippet: For immunohistochemistry on 4-μm FFPE sections, a primary antibody against pS6 240/244 (1:2,000, Cell Signaling, cat. no. 5364) was used, with an avidin–biotin peroxidase complex conjugation system (Vectastain ABC Elite, Vector laboratories) and DAB to detect the biotinylated anti-rabbit secondary antibody (1:250, Vector Laboratory, cat. no. BA-1100).

Techniques: Staining, Immunohistochemical staining, Activation Assay, Formalin-fixed Paraffin-Embedded, RNA Sequencing

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Distribution of 808 genotyped nuclei in UMAP space: 117 were classified as Mut. (pt10 = 89, pt9 = 25, pt7 = 2, pt6 = 1) or as Ref. Right, Mut. nuclei percentages per cell type (top) and across cell types (bottom). b , Representative images of co-immunofluorescence staining on formalin-fixed paraffin-embedded sections ( n = 1 per patient) showing mTOR-hyperactive (pS6 + ) neurons (NEUN + , pt2), astrocytes (GFAP + , pt2), oligodendrocytes (OLIG2 + , pt2) and microglia (IBA1 + , pt10). Nuclei (in blue) are labeled with DAPI. Scale bars, 20 µm. All patients included in this experiment are detailed in Supplementary Table . c , Cytomegalic cells representing a minor fraction of mutated cells. Left, representative immunostaining of SMI311 + DNs and VIM + BCs on frozen brain tissue from pt5. Nuclei (in blue) are labeled with DAPI for total cell counting. Scale bar, 25 µm. Right, mutated cell percentage (inferred by the detected VAF) and proportion of DNs or BCs identified in each patient ( n = 1 section/patient/staining was analyzed). d , Schematic of the distribution of mutated cells across cell types and the fraction of mutated cytomegalic cells in pt10. Astro, astrocytes; Endo, endothelial cells; Hemi, hemispherical; Oligo, oligodendrocytes; Micro, microglia.

Article Snippet: For immunohistochemistry on 4-μm FFPE sections, a primary antibody against pS6 240/244 (1:2,000, Cell Signaling, cat. no. 5364) was used, with an avidin–biotin peroxidase complex conjugation system (Vectastain ABC Elite, Vector laboratories) and DAB to detect the biotinylated anti-rabbit secondary antibody (1:250, Vector Laboratory, cat. no. BA-1100).

Techniques: Immunofluorescence, Staining, Formalin-fixed Paraffin-Embedded, Labeling, Immunostaining, Cell Counting

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Left, LCM–seq workflow for capturing pools of DNs, BCs and NNs from eight patients (pt1–5 and pt7–9). Right, heatmap of NEFM and VIM normalized expression with unsupervised hierarchical clustering. b , Label transfer of LCM–seq samples on to the snRNA-seq UMAP space showing NNs or DNs matching with GluNs and BCs with astrocytes. c , Left, NRGN and GFAP normalized expression heatmap with unsupervised hierarchical clustering. Right, co-immunofluorescence showing NRGN in pS6 + /SMI311 + DNs and GFAP in pS6 + /VIM + BCs (pt5) ( n = 1 section/patient/staining analyzed). GFAP-pS6 and VIM-pS6 double stainings were performed on two consecutive sections and the same BC was recognized in both sections. Nuclei (in blue) are labeled with DAPI. Scale bars, 50 µm. d , Visium spatial transcriptomics showing intermingled spots containing DNs and BCs across the tissue (pt5). Magnified images show representative DN- and BC-containing spots after hematoxylin and eosin staining ( n = 1 section per patient analyzed). Scale bars, 1.5 mm; insets = 55 µm. e , Top markers of DN- and BC-containing spots (pt5). Known histological markers for DNs ( NEFM ) and BCs ( CRYAB ) are enriched in spots with DNs and BCs. f , Spatial semi-supervised clustering of Visium spots showing clusters enriched in GluNs, astrocytes and oligodendrocytes (pt5) with top marker genes in parentheses. g , Distinct clusters for DNs, BCs, astrocytes (Astros) and GluNs from single cells (pt5 and pt9) of the MERSCOPE UMAP space. h , Heatmap of the top ten DN or BC markers with representative MERSCOPE images (pt5). DNs are identified as pS6 + /NEUN + and BCs as pS6 + /NEUN − ( n = 1 section per patient analyzed). Scale bars, 50 µm. i , Left, number of shared dysregulated genes across Mut. versus Ref. GluNs (snRNA-seq), DNs versus NNs (LCM–seq) and DN-containing spots (Visium). Right, top GO terms of DN upregulated genes. Ribo-nt., ribonucleotides; metab., metabolic; proc., process; Ribo-ns., ribonucleosides; RP., ribosomal proteins; rNTP, ribonucleoside triphosphates. j , Representative images of strong VDAC1 immunostaining in pS6 + DNs (pt2) ( n = 1 section/patient/staining analyzed). Scale bars, 50 µm. k , Electron microscopy of DNs (pt5) showing an accumulation of vesicular, swollen, damaged mitochondria (black circles) ( n = 1 section per patient analyzed). Scale bar, 2.5 µm. Detailed sample information for each experiment and analysis is provided in Supplementary Table . expr., expression; max., maximum; min., minimum.

Article Snippet: For immunohistochemistry on 4-μm FFPE sections, a primary antibody against pS6 240/244 (1:2,000, Cell Signaling, cat. no. 5364) was used, with an avidin–biotin peroxidase complex conjugation system (Vectastain ABC Elite, Vector laboratories) and DAB to detect the biotinylated anti-rabbit secondary antibody (1:250, Vector Laboratory, cat. no. BA-1100).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Marker, Immunostaining, Electron Microscopy

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Heatmaps showing normalized (SCTransform) gene expression levels of the 140-gene MERSCOPE panel, grouped by category. Abbreviations: DN, dysmorphic neurons; BC, balloon cells. b , Representative MERSCOPE images of top expressed genes (from Fig. ) in DN (pS6+/NEUN+) and BC (pS6+/NEUN-) from patient pt9 (n = 1 tissue section examined). Yellow puncta indicate individual transcript molecules. Scale bar = 50μm. c , Spatial detection of mitochondrial gene transcripts (yellow puncta) in DN from patients pt5 and pt9 (n = 1 tissue section examined for each patient). Scale bar = 50 μm.

Article Snippet: For immunohistochemistry on 4-μm FFPE sections, a primary antibody against pS6 240/244 (1:2,000, Cell Signaling, cat. no. 5364) was used, with an avidin–biotin peroxidase complex conjugation system (Vectastain ABC Elite, Vector laboratories) and DAB to detect the biotinylated anti-rabbit secondary antibody (1:250, Vector Laboratory, cat. no. BA-1100).

Techniques: Gene Expression

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Co-immunofluorescence analysis of VDAC1 (white) and pS6 (pink) across FCDII specimens (patients pt1-10, n = 1 section/patient). Strong VDAC1 signal is observed in dysmorphic neurons (DN, enlarged pS6+ cells, orange arrowheads), while balloon cells (BC, pS6+ cells with glassy cytoplasm, green arrowheads) display weak VDAC1 signal. Inset in pt8 shows representative BC from distinct region. Scale bar = 100 μm. b , Quantitative comparison of VDAC1 expression between DN-positive and DN-negative regions in pt2. Analysis was performed on 0.25 mm² regions of interest (ROIs, n = 4 per condition), showing percentage of VDAC1+ and pS6+ pixels. Two-sided Wilcoxon rank sum exact p-value = 0.02857*. Box plots show median, interquartile range, and min/max values. c , Quantification of VDAC1 expression between DN-positive and BC-positive regions in pt5-8. Analysis was performed on 0.25 mm 2 ROIs (n = 3 per region per patient; total n = 12 per condition). Data shown as percentage of VDAC1+ pixels and pS6+ pixels per ROI. Two-sided Wilcoxon rank sum test, exact p-value = 5.177e-06***. Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values.

Article Snippet: For immunohistochemistry on 4-μm FFPE sections, a primary antibody against pS6 240/244 (1:2,000, Cell Signaling, cat. no. 5364) was used, with an avidin–biotin peroxidase complex conjugation system (Vectastain ABC Elite, Vector laboratories) and DAB to detect the biotinylated anti-rabbit secondary antibody (1:250, Vector Laboratory, cat. no. BA-1100).

Techniques: Immunofluorescence, Comparison, Expressing

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Representative hematoxylin and eosin (HE) staining of 20μm-thick frozen brain sections showing dysmorphic neurons (DN, empty arrowheads) and balloon cells (BC, filled arrowheads) across FCDII patients (n = 1 section/patient). Scale bar: 100μm. Insets for patients pt6, pt7 and pt10 show a representative BC from distinct cortical regions. b , Immunohistochemical detection of mTOR pathway activation using phosphorylated S6 (pS6-Ser240/244) antibody on 4μm-thick formalin-fixed paraffin-embedded (FFPE) brain sections (n = 1 section/patient). DN (empty arrowheads) and BC (filled arrowheads) exhibit strong pS6 immunoreactivity. Scale bar: 50μm. Insets for patients pt7-9 show representative DN from distinct cortical regions. c , Quantification of grey and white matter proportions in 20μm-thick HE-stained frozen brain sections adjacent to tissue used for single-nucleus RNA sequencing (n = 1 section/patient).

Article Snippet: Co-immunofluorescence was performed using primary antibodies against pS6 240/244 (1:1,000, Cell Signaling, cat. no. 5364), VIM (1:100, Dako, cat. no. M0725), SMI311 (1:500, BioLegend, cat. no. 837801), GFAP (1:200, Thermo Fisher Scientific, cat. no. MA5-15086), NRGN (1:50, Thermo Fisher Scientific, cat. no. PA5-19209), OLIG2 (1:100, R&D Systems, cat. no. AF2418), IBA1 (1:500, Abcam, cat. no. ab5076), NEUN (1:500, Millipore, cat. no. MAB377) and VDAC1 (1:500, Abcam, cat. no. ab16814).

Techniques: Staining, Immunohistochemical staining, Activation Assay, Formalin-fixed Paraffin-Embedded, RNA Sequencing

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Distribution of 808 genotyped nuclei in UMAP space: 117 were classified as Mut. (pt10 = 89, pt9 = 25, pt7 = 2, pt6 = 1) or as Ref. Right, Mut. nuclei percentages per cell type (top) and across cell types (bottom). b , Representative images of co-immunofluorescence staining on formalin-fixed paraffin-embedded sections ( n = 1 per patient) showing mTOR-hyperactive (pS6 + ) neurons (NEUN + , pt2), astrocytes (GFAP + , pt2), oligodendrocytes (OLIG2 + , pt2) and microglia (IBA1 + , pt10). Nuclei (in blue) are labeled with DAPI. Scale bars, 20 µm. All patients included in this experiment are detailed in Supplementary Table . c , Cytomegalic cells representing a minor fraction of mutated cells. Left, representative immunostaining of SMI311 + DNs and VIM + BCs on frozen brain tissue from pt5. Nuclei (in blue) are labeled with DAPI for total cell counting. Scale bar, 25 µm. Right, mutated cell percentage (inferred by the detected VAF) and proportion of DNs or BCs identified in each patient ( n = 1 section/patient/staining was analyzed). d , Schematic of the distribution of mutated cells across cell types and the fraction of mutated cytomegalic cells in pt10. Astro, astrocytes; Endo, endothelial cells; Hemi, hemispherical; Oligo, oligodendrocytes; Micro, microglia.

Article Snippet: Co-immunofluorescence was performed using primary antibodies against pS6 240/244 (1:1,000, Cell Signaling, cat. no. 5364), VIM (1:100, Dako, cat. no. M0725), SMI311 (1:500, BioLegend, cat. no. 837801), GFAP (1:200, Thermo Fisher Scientific, cat. no. MA5-15086), NRGN (1:50, Thermo Fisher Scientific, cat. no. PA5-19209), OLIG2 (1:100, R&D Systems, cat. no. AF2418), IBA1 (1:500, Abcam, cat. no. ab5076), NEUN (1:500, Millipore, cat. no. MAB377) and VDAC1 (1:500, Abcam, cat. no. ab16814).

Techniques: Immunofluorescence, Staining, Formalin-fixed Paraffin-Embedded, Labeling, Immunostaining, Cell Counting

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Left, LCM–seq workflow for capturing pools of DNs, BCs and NNs from eight patients (pt1–5 and pt7–9). Right, heatmap of NEFM and VIM normalized expression with unsupervised hierarchical clustering. b , Label transfer of LCM–seq samples on to the snRNA-seq UMAP space showing NNs or DNs matching with GluNs and BCs with astrocytes. c , Left, NRGN and GFAP normalized expression heatmap with unsupervised hierarchical clustering. Right, co-immunofluorescence showing NRGN in pS6 + /SMI311 + DNs and GFAP in pS6 + /VIM + BCs (pt5) ( n = 1 section/patient/staining analyzed). GFAP-pS6 and VIM-pS6 double stainings were performed on two consecutive sections and the same BC was recognized in both sections. Nuclei (in blue) are labeled with DAPI. Scale bars, 50 µm. d , Visium spatial transcriptomics showing intermingled spots containing DNs and BCs across the tissue (pt5). Magnified images show representative DN- and BC-containing spots after hematoxylin and eosin staining ( n = 1 section per patient analyzed). Scale bars, 1.5 mm; insets = 55 µm. e , Top markers of DN- and BC-containing spots (pt5). Known histological markers for DNs ( NEFM ) and BCs ( CRYAB ) are enriched in spots with DNs and BCs. f , Spatial semi-supervised clustering of Visium spots showing clusters enriched in GluNs, astrocytes and oligodendrocytes (pt5) with top marker genes in parentheses. g , Distinct clusters for DNs, BCs, astrocytes (Astros) and GluNs from single cells (pt5 and pt9) of the MERSCOPE UMAP space. h , Heatmap of the top ten DN or BC markers with representative MERSCOPE images (pt5). DNs are identified as pS6 + /NEUN + and BCs as pS6 + /NEUN − ( n = 1 section per patient analyzed). Scale bars, 50 µm. i , Left, number of shared dysregulated genes across Mut. versus Ref. GluNs (snRNA-seq), DNs versus NNs (LCM–seq) and DN-containing spots (Visium). Right, top GO terms of DN upregulated genes. Ribo-nt., ribonucleotides; metab., metabolic; proc., process; Ribo-ns., ribonucleosides; RP., ribosomal proteins; rNTP, ribonucleoside triphosphates. j , Representative images of strong VDAC1 immunostaining in pS6 + DNs (pt2) ( n = 1 section/patient/staining analyzed). Scale bars, 50 µm. k , Electron microscopy of DNs (pt5) showing an accumulation of vesicular, swollen, damaged mitochondria (black circles) ( n = 1 section per patient analyzed). Scale bar, 2.5 µm. Detailed sample information for each experiment and analysis is provided in Supplementary Table . expr., expression; max., maximum; min., minimum.

Article Snippet: Co-immunofluorescence was performed using primary antibodies against pS6 240/244 (1:1,000, Cell Signaling, cat. no. 5364), VIM (1:100, Dako, cat. no. M0725), SMI311 (1:500, BioLegend, cat. no. 837801), GFAP (1:200, Thermo Fisher Scientific, cat. no. MA5-15086), NRGN (1:50, Thermo Fisher Scientific, cat. no. PA5-19209), OLIG2 (1:100, R&D Systems, cat. no. AF2418), IBA1 (1:500, Abcam, cat. no. ab5076), NEUN (1:500, Millipore, cat. no. MAB377) and VDAC1 (1:500, Abcam, cat. no. ab16814).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Marker, Immunostaining, Electron Microscopy

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Heatmaps showing normalized (SCTransform) gene expression levels of the 140-gene MERSCOPE panel, grouped by category. Abbreviations: DN, dysmorphic neurons; BC, balloon cells. b , Representative MERSCOPE images of top expressed genes (from Fig. ) in DN (pS6+/NEUN+) and BC (pS6+/NEUN-) from patient pt9 (n = 1 tissue section examined). Yellow puncta indicate individual transcript molecules. Scale bar = 50μm. c , Spatial detection of mitochondrial gene transcripts (yellow puncta) in DN from patients pt5 and pt9 (n = 1 tissue section examined for each patient). Scale bar = 50 μm.

Article Snippet: Co-immunofluorescence was performed using primary antibodies against pS6 240/244 (1:1,000, Cell Signaling, cat. no. 5364), VIM (1:100, Dako, cat. no. M0725), SMI311 (1:500, BioLegend, cat. no. 837801), GFAP (1:200, Thermo Fisher Scientific, cat. no. MA5-15086), NRGN (1:50, Thermo Fisher Scientific, cat. no. PA5-19209), OLIG2 (1:100, R&D Systems, cat. no. AF2418), IBA1 (1:500, Abcam, cat. no. ab5076), NEUN (1:500, Millipore, cat. no. MAB377) and VDAC1 (1:500, Abcam, cat. no. ab16814).

Techniques: Gene Expression

Journal: Nature Neuroscience

Article Title: Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia

doi: 10.1038/s41593-025-01936-z

Figure Lengend Snippet: a , Co-immunofluorescence analysis of VDAC1 (white) and pS6 (pink) across FCDII specimens (patients pt1-10, n = 1 section/patient). Strong VDAC1 signal is observed in dysmorphic neurons (DN, enlarged pS6+ cells, orange arrowheads), while balloon cells (BC, pS6+ cells with glassy cytoplasm, green arrowheads) display weak VDAC1 signal. Inset in pt8 shows representative BC from distinct region. Scale bar = 100 μm. b , Quantitative comparison of VDAC1 expression between DN-positive and DN-negative regions in pt2. Analysis was performed on 0.25 mm² regions of interest (ROIs, n = 4 per condition), showing percentage of VDAC1+ and pS6+ pixels. Two-sided Wilcoxon rank sum exact p-value = 0.02857*. Box plots show median, interquartile range, and min/max values. c , Quantification of VDAC1 expression between DN-positive and BC-positive regions in pt5-8. Analysis was performed on 0.25 mm 2 ROIs (n = 3 per region per patient; total n = 12 per condition). Data shown as percentage of VDAC1+ pixels and pS6+ pixels per ROI. Two-sided Wilcoxon rank sum test, exact p-value = 5.177e-06***. Box plots depict the median and interquartile range, with whiskers indicating minimum and maximum values.

Article Snippet: Co-immunofluorescence was performed using primary antibodies against pS6 240/244 (1:1,000, Cell Signaling, cat. no. 5364), VIM (1:100, Dako, cat. no. M0725), SMI311 (1:500, BioLegend, cat. no. 837801), GFAP (1:200, Thermo Fisher Scientific, cat. no. MA5-15086), NRGN (1:50, Thermo Fisher Scientific, cat. no. PA5-19209), OLIG2 (1:100, R&D Systems, cat. no. AF2418), IBA1 (1:500, Abcam, cat. no. ab5076), NEUN (1:500, Millipore, cat. no. MAB377) and VDAC1 (1:500, Abcam, cat. no. ab16814).

Techniques: Immunofluorescence, Comparison, Expressing