sheep anti tbr2  (R&D Systems)

Journal: Nature

Article Title: Dysregulation of mTOR signalling is a converging mechanism in lissencephaly

doi: 10.1038/s41586-024-08341-9

Figure Lengend Snippet: a , Control, patient, knock-in, and rescue organoids at D50 immunostained for apoptotic cell marker cleaved-caspase 3 (CC3). Dashed yellow lines delineate the SVZ/VZ. b , Quantification of CC3 + cells per mm 2 SVZ/VZ area. c , Control, patient, knock-in, and rescue organoids at D50 immunostained for oRG cell marker HOPX. Dashed yellow lines delineate the SVZ/VZ. d , Quantification of HOPX + cells per mm 2 SVZ/VZ area. e , Control, patient, knock-in, and rescue organoids at D70 immunostained for IPC marker TBR2. Dashed yellow lines delineate the SVZ. f , Quantification of TBR2 + cells per mm 2 SVZ area. g , Control, patient, knock-in, and rescue organoids at D70 immunostained for oRG marker HOPX, TBR2, and CC3. h , i , Quantification of the faction of CC3 + cells co-expressing either TBR2 ( h ) or HOPX ( i ). n = 12 cortical regions, n = 6 organoids, 2 batches of 3 organoids per genotype ( b, d, f ); n = 6 cortical regions, n = 6 organoids, 2 batches per genotype ( h, I ). Statistical test: one-way ANOVA; ****, P < 0.0001; **, P < 0.01; *, P < 0.05; ns, not significant. Scale bars, 100 µm ( a, c, e ) and 50 µm ( g ).

Article Snippet: The following primary antibodies were used: mouse anti-MAP2 (1:500; MAB3418, Millipore); mouse anti-SOX2 (1:500; sc-365823; Santa Cruz); rat anti-SOX2 (1:500; 14-9811-82, Invitrogen); rat anti-CTIP2 (1:500; ab18465; Abcam); rabbit anti-SATB2 (1:500; ab34735, Abcam); rabbit anti-TBR1 (1:500; ab31940, Abcam); rabbit anti-HOPX (1:2,500; HPA030180, Sigma); mouse anti-HOPX (1:250; sc-398703); rabbit anti-CC3 (1:1,000; 9661, Cell Signaling Technologies); mouse anti-PH3 (1:500; 9706, Cell Signaling Technologies); mouse anti-Ki67 (1:500; 556003, BD Biosciences); rabbit anti-EOMES (TBR2) (1:500; HPA028896, Sigma); sheep anti-EOMES (TBR2) (1:200; AF6166, R&D Systems); and rabbit anti-phospho-S6 ribosomal protein (pS6) (1:200; 4858, Cell Signaling Technologies).

Techniques: Control, Knock-In, Marker, Expressing

Journal: Nature

Article Title: Dysregulation of mTOR signalling is a converging mechanism in lissencephaly

doi: 10.1038/s41586-024-08341-9

Figure Lengend Snippet: a - b Control organoids at D70 immunostained for pS6 and oRG marker HOPX or IPC marker TBR2. Nuclei are stained with DAPI. Dashed white box indicates the magnified area shown to the right; dashed white lines delineate the SVZ/VZ. c , Quantification of HOPX + /pS6 + or TBR2 + /pS6 + cells in the SVZ. d , Control, patient, knock-in, and rescue organoids at D70 immunostained for SOX2, CTIP2, and pS6. e , Quantification of pS6 + /SOX2 + cells per mm 2 SVZ area. f , Quantification of pS6 relative intensity in the SVZ area. n = 13 cortical regions, n = 6 organoids, 2 batches of 3 organoids per genotype ( c ); n = 8 cortical regions, n = 4 organoids, 2 batches of 2 organoids per genotype ( e, f ). Statistical tests: two-tailed unpaired t-test; ****, P < 0.0001 (c) ; one-way ANOVA; ****, P < 0.0001; **, P < 0.01; ns, not significant (e, f) . Data are mean±s.d. ( c, e, f ). Scale bars, 100 µm.

Article Snippet: The following primary antibodies were used: mouse anti-MAP2 (1:500; MAB3418, Millipore); mouse anti-SOX2 (1:500; sc-365823; Santa Cruz); rat anti-SOX2 (1:500; 14-9811-82, Invitrogen); rat anti-CTIP2 (1:500; ab18465; Abcam); rabbit anti-SATB2 (1:500; ab34735, Abcam); rabbit anti-TBR1 (1:500; ab31940, Abcam); rabbit anti-HOPX (1:2,500; HPA030180, Sigma); mouse anti-HOPX (1:250; sc-398703); rabbit anti-CC3 (1:1,000; 9661, Cell Signaling Technologies); mouse anti-PH3 (1:500; 9706, Cell Signaling Technologies); mouse anti-Ki67 (1:500; 556003, BD Biosciences); rabbit anti-EOMES (TBR2) (1:500; HPA028896, Sigma); sheep anti-EOMES (TBR2) (1:200; AF6166, R&D Systems); and rabbit anti-phospho-S6 ribosomal protein (pS6) (1:200; 4858, Cell Signaling Technologies).

Techniques: Control, Marker, Staining, Knock-In, Two Tailed Test