rat anti tbr2  (Proteintech)

Journal: The EMBO Journal

Article Title: Kinesin-like motor protein KIF23 maintains neural stem and progenitor cell pools in the developing cortex

doi: 10.1038/s44318-024-00327-7

Figure Lengend Snippet: ( A – C ) Representative images of E15.5 control and Kif23- KD cortices stained against GFP, CC3, and DAPI showing pyknotic cells across various cortical zones ( A ). Ventricular zone (VZ), subventricular zone (SVZ), and intermediate zone (IZ). Scale bars, 20 μm. Quantification of the percentage of pyknotic cells VZ, SVZ, and IZ, respectively ( B ) or CC3 + cells ( C ) relative to the total GFP + cells within the 320 μm wide column of E15.5 cortices. The data represent the mean ± SD (Control n = 1167 cells, 3 embryos; Kif23 -KD n = 1248 cells, embryos). Multiple t tests, p values from left to right: *** p = 0.000294, ** p = 0.005134, ** p = 0.003350 ( B ). Two-tailed Student’s t test, **** p = 6.3E-05 ( C ). ( D , E ) Representative images of E15.5 Kif23- KD cortical sections stained for GFP, Sox2, Tbr2, Hu, Tuj1, and DAPI ( D ). Scale bars, 20 μm. Close arrowheads indicate Sox2 + , or Tbr2 + , or Hu + , or Tuj1 + pyknotic cells. Open arrowheads indicate Sox2 - , or Tbr2 - , or Hu - , or Tuj1 - pyknotic cells. Quantification of the ratio of pyknotic cells positive for Sox2, Tbr2, Hu, and Tuj1 within the 110 μm wide column of E15.5 cortices ( E ). The data represent the mean ± SD ( n = 148 cells, 3 embryos for Sox2 + group; n = 131 cells, 3 embryos for Tbr2 + group; n = 163 cells, 4 embryos for Hu + group; n = 234 cells, 4 embryos for Tuj1 + group). ( F , G ) Representative images of E15.5 Kif23 siRNA transfected cortices stained for CC3 and DAPI ( F ). Top right panel: binucleated cell stained with DAPI. Bottom right panel: the 3D image of the binucleated cell. Scale bars, 20 μm (left) and 1 μm (right). Open arrowheads indicate pyknotic single with micronuclei. Closed arrowheads indicate pyknotic doublets. Quantification of the percentage of pyknotic cells relative to the total GFP + cells within the 320 μm wide column of E15.5 cortices ( G ). The data represent the mean ± SD (Control n = 1167 cells, 3 embryos; Kif23 -KD n = 1248 cells, 3 embryos). Multiple t tests, p values from left to right: *** p = 0.000193, *** p = 0.000302. .

Article Snippet: Rat anti-Tbr2 (1:1000) , Invitrogen , Cat# 14487582 RRID:AB_11042577.

Techniques: Control, Staining, Two Tailed Test, Transfection

Journal: The EMBO Journal

Article Title: Kinesin-like motor protein KIF23 maintains neural stem and progenitor cell pools in the developing cortex

doi: 10.1038/s44318-024-00327-7

Figure Lengend Snippet: ( A , B ) Representative images of the control and Kif23- KD cortices at E15.5 stained for GFP and Pax6 ( A ). Boxed areas denote zoomed areas. Scale bars, 20 μm. Quantification of the percentage of Pax6 + GFP + cells relative to the total GFP + cells within the 100 μm wide column of E15.5 cortices ( B ). The data represent the mean ± SD (Control n = 524 cells, 3 embryos; Kif23 -KD n = 450 cells, 3 embryos). Two-tailed Student’s t test, ** p = 0.006541. ( C , D ) Representative images of the control and Kif23- KD cortices at E15.5 stained for GFP and Tbr2 ( C ). Boxed areas denote zoomed areas. Scale bars, 20 μm. Quantification of the percentage of Tbr2 + GFP + cells relative to the total GFP + cells within the 100 μm wide column of E15.5 cortices ( D ). The data represent the mean ± SD (Control n = 547 cells, 3 embryos; Kif23 -KD n = 494 cells, 3 embryos). Two-tailed Student’s t test, ** p = 0.003548. ( E , F ) Timeline of the in-utero electroporation and EdU injection (left). Electroporated cortical sections were stained for GFP and EdU (right) ( E ). Dashed lines illustrate the boundary for the ventricular zone (VZ). Boxed areas denote zoomed areas. Scattered lines encircle GFP + cells that are EdU + or EdU − . Scale bars, 20 μm. Quantification of the percentage of EdU + GFP + cells relative to the total GFP + cells within the VZ of 150 μm wide column of E16.5 cortices ( F ). The data represent the mean ± SD (Control n = 415 cells, 3 embryos; Kif23 -KD n = 87 cells, 3 embryos). Two-tailed Student’s t test, **** p = 7.8E-05. ( G , H ) Representative images of the control and Kif23- KD cortices at E16.5 stained for GFP and PH3 ( G ). Dashed lines illustrate the boundary for the ventricular zone (VZ). Boxed areas denote zoomed areas. Scale bars, 25 μm. Quantification of the percentage of PH3 + GFP + cells relative to the total GFP + cells within the VZ of 200 μm wide column of E16.5 cortices ( H ). The data represent the mean ± SD (Control n = 208 cells, 3 embryos; Kif23 -KD n = 72 cells, 3 embryos). Two-tailed Student’s t test, * p = 0.017095. ( I , J ) Timeline of the in-utero electroporation and EdU injection (left). Electroporated cortical sections were stained for GFP, Ki67, and EdU (right) ( I ). Boxed areas denote zoomed areas. Scattered lines encircle EdU + GFP + cells that are Ki67 + or Ki67 − . Scale bars, 20 μm. Quantification of the percentage of EdU + GFP + Ki67 − cells relative to the total EdU + GFP + cells within the 200 μm wide column of E16.5 cortices ( J ). The data represent the mean ± SD (Control n = 460 cells, 4 embryos; Kif23 -KD n = 121 cells, 4 embryos). Two-tailed Student’s t test, *** p = 0.000318. .

Article Snippet: Rat anti-Tbr2 (1:1000) , Invitrogen , Cat# 14487582 RRID:AB_11042577.

Techniques: Control, Staining, Two Tailed Test, In Utero, Electroporation, Injection

Journal: The EMBO Journal

Article Title: Kinesin-like motor protein KIF23 maintains neural stem and progenitor cell pools in the developing cortex

doi: 10.1038/s44318-024-00327-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rat anti-Tbr2 (1:1000) , Invitrogen , Cat# 14487582 RRID:AB_11042577.

Techniques: Recombinant, Sequencing, Control, Amplification, Clone Assay, Plasmid Preparation, Labeling, Imaging, Software, Microscopy

Journal: bioRxiv

Article Title: AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

doi: 10.1101/2024.04.15.589462

Figure Lengend Snippet: (A) Immunostaining for PAX6 (green) and PH3 (magenta) in E15.5 WT and Auts2 del8/del8 cerebral cortices. The graph shows the number of PAX6 + and PH3 + cells on the ventricular surface in WT, Auts2 del8/+ , and Auts2 del8/del8 mice. (B) Immunostaining for TBR2 (green) and PH3 (magenta) in E15.5 WT and Auts2 del8/del8 cerebral cortices. The graph shows the number of TBR2 + and PH3 + cells in WT, Auts2 del8/+ , and Auts2 del8/del8 mice. (C–F) EdU was administered intraperitoneally to pregnant mice 30 min before sacrifice at E15.5. (C) Representative images of staining for PAX6 (green) and EdU (magenta) in WT and Auts2 del8/del8 cortical sections. (D) Representative images of staining for TBR2 (green), KI67 (magenta), and EdU (white) in WT and Auts2 del8/del8 cortical sections. (E, F) The graphs show the percentage of EdU + cells among PAX6 + cells (E) or TBR2 + KI67 + cells (F) in WT, Auts2 del8/+ and Auts2 del8/del8 mice. VZ, ventricular zone; SVZ, subeventricular zone; IZ, intermediate zone. (G) The cell-cycle length estimate in TBR2 + cells using the BrdU and EdU double-labeling method at E15.5. BrdU and EdU are administered to pregnant mice at 2 h and 30 min before sacrifice. Ts, S-phase length; Tc, cell cycle length; L cells , BrdU + , and EdU-negative cells; S cells , EdU + cells; P cells , TBR2 + cells. The number of cells was quantified at the rostral point. Data are presented as the mean ± SD (N = three mice, six sections). NS, not significant, One-way ANOVA with Dunnett’s post-hoc test. Scale bars, 50 µm.

Article Snippet: The following primary antibodies were used, rabbit anti-CUX1 (1:1000; sc-13024; Santa Cruz Biotechnology, Danvers, MA), rat anti-CTIP2 (1:3000; ab18465; abcam), rabbit anti-cleaved caspase-3 (1:400; 9661S; Cell Signaling Technology, Danvers, MA), rabbit anti-PAX6 (1:500; 901301; BioLegend, San Diego, CA), rabbit anti-TBR2 (1:1000; ab183991; abcam), rat anti-TBR2 (1:500; 14-4876-80; Invitrogen, Carlsbad, CA), rat anti-PH3 (pSer28) (1:400; H9908; Sigma-Aldrich, St. Louis, MO), rat anti-KI67 (1:500; 14-5698-82; Invitrogen), sheep anti-BrdU (1:200; ab1893; abcam), chicken anti-GFP (1:1000; ab13970; abcam), and goat anti-SOX2 (1:200; AF2018; R&D systems, Minneapolis, MN).

Techniques: Immunostaining, Staining, Labeling

Journal: bioRxiv

Article Title: AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

doi: 10.1101/2024.04.15.589462

Figure Lengend Snippet: (A) Representative images of immunostaining for PAX6 in WT and Auts2 del8/del8 cortical sections at E15.5. The graph shows the number of PAX6 + cells within a 100 µm wide field. (B) Representative images of immunostaining for TBR2 (green) and KI67 (magenta) in WT and Auts2 del8/del8 cortical sections at E15.5. The graph shows the number of TBR2 + cells within a 100 µm wide field (top) and the ratio of KI67 + cells in TBR2 + cells (bottom) (C–H) EdU was administered intraperitoneally to pregnant mice 30 min before sacrifice at E12.5. (D) Representative staining images for PAX6 (green) and EdU (magenta) in WT and Auts2 del8/del8 cortical sections. (E) Representative staining images for TBR2 (green) and EdU (magenta) in WT and Auts2 del8/del8 cortical sections. (E, G) The graphs show the number of PAX6 + (E) and TBR2 + (G) cells within a 100 µm wide field. (F, H) The graphs show the percentage of EdU + cells in PAX6 + cells (F) and TBR2 + cells (H). (I) Representative Immunostaining images for PAX6 (green) and PH3 (magenta) in WT and Auts2 del8/del8 cortical sections at E12.5. The graph shows the number of PAX6 + and PH3 + cells on the ventricular surface in WT, Auts2 del8/+ , and Auts2 del8/del8 mice. (J) Representative Immunostaining images for TBR2 (green) and PH3 (magenta) in WT and Auts2 del8/del8 cortical sections at E12.5. The graph shows the number of TBR2 + and PH3 + cells in WT, Auts2 del8/+ , and Auts2 del8/del8 mice. Arrows indicate TBR2 + and PH3 + cells. The number of cells was quantified at the rostral point. Data are presented as the mean ± SD (N = three mice, six sections). NS, not significant, One-way ANOVA with Dunnett’s post-hoc test. Scale bars, 50 µm. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone.

Article Snippet: The following primary antibodies were used, rabbit anti-CUX1 (1:1000; sc-13024; Santa Cruz Biotechnology, Danvers, MA), rat anti-CTIP2 (1:3000; ab18465; abcam), rabbit anti-cleaved caspase-3 (1:400; 9661S; Cell Signaling Technology, Danvers, MA), rabbit anti-PAX6 (1:500; 901301; BioLegend, San Diego, CA), rabbit anti-TBR2 (1:1000; ab183991; abcam), rat anti-TBR2 (1:500; 14-4876-80; Invitrogen, Carlsbad, CA), rat anti-PH3 (pSer28) (1:400; H9908; Sigma-Aldrich, St. Louis, MO), rat anti-KI67 (1:500; 14-5698-82; Invitrogen), sheep anti-BrdU (1:200; ab1893; abcam), chicken anti-GFP (1:1000; ab13970; abcam), and goat anti-SOX2 (1:200; AF2018; R&D systems, Minneapolis, MN).

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

doi: 10.1101/2024.04.15.589462

Figure Lengend Snippet: (A) Targeting strategy of CRISPR/Cas9 method for the generation of Eomes T2A-d2EGFP knock-in mouse. The DNA sequences for T2A-d2EGFP were inserted between the last amino acid and the stop codon of the Eomes locus. L-HA, left homology arm; R-HA, right homology arm; ssDNA, single-stranded donor DNA; F, forward primer for genotyping; R1 and R2, reverse primers for genotyping. (B) In vitro digestion assay to check the cleavage activity of the crRNA designed in (A). Targeted PCR products of the Eomes locus were cleaved in the presence of the chemically synthesized guide RNA ( Eomes crRNA/tracrRNA) combined with Cas9 protein. M, molecular marker. (C) Genotyping for WT and Eomes T2A-d2EGFP/+ (KI) mouse using primers indicated in (A). (D) Western blotting analysis of cerebral cortical lysates in WT, Eomes T2A-d2EGFP/+ (KI/+), and Eomes T2A-d2EGFP/T2A-d2EGFP (KI/KI) mice at E14.5 with anti-TBR2 and anti-ß-actin antibodies. A very small amount of uncleaved TBR2-T2A-d2EGFP protein was observed at the long exposure (arrow); however, most signals for TBR2 were detected around the expected size for TBR2 protein (short exposure), indicating that TBR2 and d2EGFP proteins were efficiently cleaved in those mice. (E) Whole-mount images in WT and Eomes T2A-d2EGFP/T2A-d2EGFP (KI/KI) mouse brains at E18.5. Scale bars, 1 mm. (F) Representative images of staining with DAPI (blue), anti-GFP (green), and anti-TBR2 (magenta) antibodies in Eomes T2A-d2EGFP/+ cortical sections at E14.5. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars, 50 µm. (G) RT-qPCR analysis for Sox2, Nes, Pax6, Eomes, Neurod1 and Tbr1 in the cerebral cortex, sorted CD133 high cells and sorted EGFP + cells from Eomes T2A-d2EGFP/+ mice at E15.5. Data are presented as mean ± SD (N = four biological replicates); One-way ANOVA with Turkey’s post-hoc test or Kruskal–Wallis test.

Article Snippet: The following primary antibodies were used, rabbit anti-CUX1 (1:1000; sc-13024; Santa Cruz Biotechnology, Danvers, MA), rat anti-CTIP2 (1:3000; ab18465; abcam), rabbit anti-cleaved caspase-3 (1:400; 9661S; Cell Signaling Technology, Danvers, MA), rabbit anti-PAX6 (1:500; 901301; BioLegend, San Diego, CA), rabbit anti-TBR2 (1:1000; ab183991; abcam), rat anti-TBR2 (1:500; 14-4876-80; Invitrogen, Carlsbad, CA), rat anti-PH3 (pSer28) (1:400; H9908; Sigma-Aldrich, St. Louis, MO), rat anti-KI67 (1:500; 14-5698-82; Invitrogen), sheep anti-BrdU (1:200; ab1893; abcam), chicken anti-GFP (1:1000; ab13970; abcam), and goat anti-SOX2 (1:200; AF2018; R&D systems, Minneapolis, MN).

Techniques: CRISPR, Knock-In, In Vitro, Activity Assay, Synthesized, Marker, Western Blot, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

doi: 10.1101/2024.04.15.589462

Figure Lengend Snippet: (A) Experimental design for transcriptional analysis. CD133 high and EGFP + cells were sorted from Eomes T2A-d2EGFP/+ ; Auts2 flox/flox (Control) or Eomes T2A-d2EGFP/+ ; Emx1 Cre/+ ; Auts2 flox/flox ( Auts2 cKO) cortices at E15.5. (B) Representative plot showing sorting gates for CD133 high and EGFP + cells. (C) Expression levels of Nes, Sox2, Pax6, Hes1, Eomes, Neurod1, Tubb3, Tbr1, Fezf2, Auts2, Rpl18, and Rplp1 transcripts in sorted each cell type from E15.5 control mouse cortex. Rpl18 and Rplp1 are presented as housekeeping genes. TPM, Transcripts per million. Adjusted P-value calculated by DESeq2 was shown. (D) Immunostaining of CD133 high and EGFP + cells sorted by FACS from E15.5 Eomes T2A-d2EGFP/+ cortex with DAPI (blue), anti-SOX2 (magenta) and anti-TBR2 (cyan) antibodies. Scale bars, 20 µm. (E) Volcano plot showing differences in gene expression in EGFP + cells between control and Auts2 cKO mice. Red dots indicate upregulated and downregulated genes (P-value < 0.01 and |Log 2 fold-change| > 0.58). (F) Heatmap for the differential expressed genes (DEG) RNA levels in EGFP + cells from control and Auts2 cKO mice. The color scale is shown on the left bottom. (G) DAVID Gene Ontology biological process analysis of upregulated genes in EGFP + cells. The top 10 GO terms with Benjamini-Hochberg adjusted P-value are shown. Numbers in parentheses indicate the gene count. (H) RT-qPCR analysis for Bhlhe22, Cadm1, Efna5, Robo1, Slit1, and Zic2 in sorted EGFP + cells from control and Auts2 cKO mice at E15.5. Data are presented as the mean ± SD (N=four mice); unpaired Student’s t-test. All transcriptome analyses used six biological replicates per genotype.

Article Snippet: The following primary antibodies were used, rabbit anti-CUX1 (1:1000; sc-13024; Santa Cruz Biotechnology, Danvers, MA), rat anti-CTIP2 (1:3000; ab18465; abcam), rabbit anti-cleaved caspase-3 (1:400; 9661S; Cell Signaling Technology, Danvers, MA), rabbit anti-PAX6 (1:500; 901301; BioLegend, San Diego, CA), rabbit anti-TBR2 (1:1000; ab183991; abcam), rat anti-TBR2 (1:500; 14-4876-80; Invitrogen, Carlsbad, CA), rat anti-PH3 (pSer28) (1:400; H9908; Sigma-Aldrich, St. Louis, MO), rat anti-KI67 (1:500; 14-5698-82; Invitrogen), sheep anti-BrdU (1:200; ab1893; abcam), chicken anti-GFP (1:1000; ab13970; abcam), and goat anti-SOX2 (1:200; AF2018; R&D systems, Minneapolis, MN).

Techniques: Expressing, Immunostaining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

doi: 10.1101/2024.04.15.589462

Figure Lengend Snippet: (A) In utero electroporation (IUE) of either an empty vector (control) or a Robo1 expression vector ( Robo1 OE) together with a H3-GFP expression vector was performed into WT cortices at E13.5. The cortical sections are stained with DAPI (blue), anti-GFP (green), anti-PH3 (magenta), and anti-TBR2 (white) antibodies at E15.5. The graph shows the ratio of PH3 + cells in TBR2 + and GFP + cells. Arrows indicate GFP + , PH3 + , and TBR2 + cells. (B) IUE of an empty (control) or a Robo1 expression vector ( Robo1 OE) and a H3-GFP expression vector into WT cortices at E14.5. The cortical sections are immunostained with anti-GFP (green), anti-KI67 (magenta), and anti-TBR2 (white) antibodies at E16.5. The graph shows the proportion of KI67 + and TBR2-negative cells located in the VZ (RGCs), TBR2 + and KI67 + cells (IPCs), and KI67-negative differentiated cells (postmitotic neurons) among total GFP + cells. The percentage of RGCs and IPCs in electroporated cells was not different between Robo1 OE and control. (C) Auts2 flox/flox or Emx1 Cre/+ ; Auts2 flox/flox ( Auts2 cKO) cortices were electroporated with scrambled or Robo1 shRNA vector in utero at E13.5 and immunostained with anti-GFP (green), anti-PH3 (magenta) and anti-TBR2 (white) antibodies at E15.5. The graph shows the percentage of PH3 + cells among TBR2 + and GFP + cells. Arrows indicate GFP + , PH3 + , and TBR2 + cells. Data are presented as the mean ± SD (N = three mice, six sections). NS, not significant, unpaired Student’s t-test (A, B) and One-way ANOVA with Turkey’s post-hoc test (C). Scale bars, 20 µm (A, C) and 50 µm (B).

Article Snippet: The following primary antibodies were used, rabbit anti-CUX1 (1:1000; sc-13024; Santa Cruz Biotechnology, Danvers, MA), rat anti-CTIP2 (1:3000; ab18465; abcam), rabbit anti-cleaved caspase-3 (1:400; 9661S; Cell Signaling Technology, Danvers, MA), rabbit anti-PAX6 (1:500; 901301; BioLegend, San Diego, CA), rabbit anti-TBR2 (1:1000; ab183991; abcam), rat anti-TBR2 (1:500; 14-4876-80; Invitrogen, Carlsbad, CA), rat anti-PH3 (pSer28) (1:400; H9908; Sigma-Aldrich, St. Louis, MO), rat anti-KI67 (1:500; 14-5698-82; Invitrogen), sheep anti-BrdU (1:200; ab1893; abcam), chicken anti-GFP (1:1000; ab13970; abcam), and goat anti-SOX2 (1:200; AF2018; R&D systems, Minneapolis, MN).

Techniques: In Utero, Electroporation, Plasmid Preparation, Expressing, Staining, shRNA

Journal: bioRxiv

Article Title: AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

doi: 10.1101/2024.04.15.589462

Figure Lengend Snippet: (A) In vitro knockdown (KD) experiment of ROBO1 expression by co-transfection of a Robo1 expression vector and scrambled shRNA or Robo1 shRNA vectors into HEK293 cells. The cell lysates were immunoblotted with anti-ROBO1 and anti-ϕ3-actin antibodies 2 days after transfection. (B) In utero electroporation (IUE) of scrambled or Robo1 shRNA into Auts2 flox/flox or Emx1 Cre/+ ; Auts2 flox/flox ( Auts2 cKO) cortices at E14.5, followed by immunostaining with anti-GFP (green), anti-KI67 (magenta) and anti-TBR2 (white) at E16.5. The graph shows the proportion of KI67 + and TBR2-negative cells located in the VZ (RGCs), TBR2 + and KI67 + cells (IPCs), and KI67-negative cells (postmitotic neurons) among total GFP + cells. The percentage of RGCs and IPCs in electroporated cells was not different among the indicated samples. Data are presented as the mean ± SD (N = three mice, six sections). NS, not significant, One-way ANOVA with Dunnett’s post-hoc test. Scale bars, 50 µm.

Article Snippet: The following primary antibodies were used, rabbit anti-CUX1 (1:1000; sc-13024; Santa Cruz Biotechnology, Danvers, MA), rat anti-CTIP2 (1:3000; ab18465; abcam), rabbit anti-cleaved caspase-3 (1:400; 9661S; Cell Signaling Technology, Danvers, MA), rabbit anti-PAX6 (1:500; 901301; BioLegend, San Diego, CA), rabbit anti-TBR2 (1:1000; ab183991; abcam), rat anti-TBR2 (1:500; 14-4876-80; Invitrogen, Carlsbad, CA), rat anti-PH3 (pSer28) (1:400; H9908; Sigma-Aldrich, St. Louis, MO), rat anti-KI67 (1:500; 14-5698-82; Invitrogen), sheep anti-BrdU (1:200; ab1893; abcam), chicken anti-GFP (1:1000; ab13970; abcam), and goat anti-SOX2 (1:200; AF2018; R&D systems, Minneapolis, MN).

Techniques: In Vitro, Expressing, Cotransfection, Plasmid Preparation, shRNA, Transfection, In Utero, Electroporation, Immunostaining

Journal: bioRxiv

Article Title: AUTS2, a causative gene for microcephaly, regulates division of intermediate progenitor cells and production of upper-layer neurons

doi: 10.1101/2024.04.15.589462

Figure Lengend Snippet: (A) Co-immunoprecipitation assay using Neuro2a cells. 3xFLAG-tagged FL-AUTS2 expression vector or 3xFlag-tagged empty vector was transfected into Neuro2a cells. The nuclear lysates were incubated with an anti-FLAG antibody. Western blotting was performed with anti-EZH, anti-SUZ12, and anti-FLAG antibodies. (B) Co-immunoprecipitation assay using E14.5 cerebral cortices. Rabbit anti-AUTS2 and rabbit control IgG antibodies were used for immunoprecipitation. The precipitates were immunoblotted with anti-EZH2, anti-SUZ12, and anti-AUTS2 antibodies. (C) Proximity ligation assay (PLA) with rabbit anti-AUTS2 and mouse anti-EZH2 antibodies in sorted EGFP + cells from E15.5 WT cortices. Representative images show the PLA signals (red) and DAPI (blue) in cells incubated with both antibodies (top) or a single antibody (only anti-EZH2, bottom). The graph shows the number of puncta in the nucleus of each sample. N = three biological replicates, 28–35 cells. (D) Venn diagrams showing the extent of overlap between AUTS2 peaks and EZH2 (top) or SUZ12 (bottom) peaks. (E) Density profiles show the occupancy of EZH2 (top) and SUZ12 (bottom) in control and Auts2 cKO cells centered on IPC-upregulated-TSSs (±3 kb). Graphs show the enrichment of EZH2 and SUZ12 on IPC-upregulated-TSSs. N = three biological replicates. (F) IUE of indicated vectors and an H3-GFP expression vector were performed into WT cortices at E13.5. The cortical sections are stained for GFP (green), PH3 (magenta), and anti-TBR2 (white) antibodies at E15.5. The graph shows the ratio of PH3 + cells in TBR2 + and GFP + cells. Red rectangles indicate the location of the images below. Arrowheads indicate GFP + , PH3 + , and TBR2 + cells. N = five mice, 10 sections. Data are presented as the median (C) and the mean ± SD (E, F). NS, not significant, Kruskal–Wallis test (C), unpaired Student’s t-test (E), and One-way ANOVA with Dunnett’s post-hoc test (F). Scale bars, 5 µm (C), 50 µm (F).

Article Snippet: The following primary antibodies were used, rabbit anti-CUX1 (1:1000; sc-13024; Santa Cruz Biotechnology, Danvers, MA), rat anti-CTIP2 (1:3000; ab18465; abcam), rabbit anti-cleaved caspase-3 (1:400; 9661S; Cell Signaling Technology, Danvers, MA), rabbit anti-PAX6 (1:500; 901301; BioLegend, San Diego, CA), rabbit anti-TBR2 (1:1000; ab183991; abcam), rat anti-TBR2 (1:500; 14-4876-80; Invitrogen, Carlsbad, CA), rat anti-PH3 (pSer28) (1:400; H9908; Sigma-Aldrich, St. Louis, MO), rat anti-KI67 (1:500; 14-5698-82; Invitrogen), sheep anti-BrdU (1:200; ab1893; abcam), chicken anti-GFP (1:1000; ab13970; abcam), and goat anti-SOX2 (1:200; AF2018; R&D systems, Minneapolis, MN).

Techniques: Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Transfection, Incubation, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Staining

Journal: eLife

Article Title: Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

doi: 10.7554/eLife.92342

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-EOMES (Tbr2) Rat IgG2A , Invitrogen , 14-4875-82 RRID: AB_11042577 , IF(1:400).

Techniques: Recombinant, Plasmid Preparation, Sequencing, BIA-KA, SYBR Green Assay, Labeling, Clone Assay, Mutagenesis, Software, Protease Inhibitor, Magnetic Beads, Membrane, Western Blot, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Chromatography, Laser-Scanning Microscopy, Fluorescence, Real-time Polymerase Chain Reaction, Mass Spectrometry