Fig. 1

In situ hybridization of Nf1 in mouse and human brain. a Representative merged image of triple fluorescent in situ hybridization probed for Nf1 (red), αCamkII (green) and vGAT (white) in hippocampal CA1 region. Higher-magnification images of the boxed area in (a) were also shown. White arrows indicate double-positive cells for Nf1 and vGAT. Sections were also stained with DAPI (blue). In situ hybridization was performed following the manufacturers’ manual (RNAscope Multiplex Fluorescent Reagent Kit, Advanced Cell Diagnostics) and the following probes (Advanced Cell Diagnostics) were used: mouse Nf1, catalog #417351; mouse Slc32a1-C2, #319191-C2; mouse CamK2-C3, #445231-C3. Images were acquired by using Axio scan Z1 (Zeiss) and analyzed by using ImageJ (NIH). Scale bar, 10 μm. b Average particle size in αCamkII⁺ neurons or vGAT⁺ neurons in CA1. Data were collected from 627 αCamkII⁺ cells and 76 vGAT⁺ cells in hippocampal CA1 area. c Representative merged image of triple fluorescent in situ hybridization probed for Nf1 (red), αCamkII (green) and vGAT (white) in the perietal cortex. Scale bar, 10 μm. d Average particle size in αCaMKII⁺ neurons or vGAT⁺ neurons in the cortex. Data were collected from 127 αCamkII⁺ cells and 74 vGAT⁺. Data is expressed as means ± SEM. Unpaired t-test, ****p < 0.0001. e, g Representative merged image of duplex chromogenic in situ hybridization probed for NF1 (blue) and αCaMKII (red) or NF1 (blue), vGAT (red) and hematoxylin for counter-staining (light-purple color) in human cortex [e, sample #20399, 3 years old female diagnosed with focal cortical dysplasia type I (temporal cortex); g sample #17490, 2 years old male diagnosed with focal cortical dysplasia type I (frontal cortex)]. Black arrows indicate co-stained cells for either NF1 and αCaMKII or NF1 and vGAT. Chromogenic detection methods according to the manufacture’s instruction (RNAscope duplex chromogenic detection Kit, Advanced Cell Diagnostics). Gene specific probes for human NF1, αCAMKII, and vGAT (human NF1, catalog # 419731; human SLC32A1-C2, #415681-C2; human CAMK2-C2, #521261-C2) were used. Images were acquired by using Aperio scan (Leica Biosystems) and analyzed by using ImageJ (NIH). Images were separated into 3 determined colors (red, green and blue) by ‘colour deconvolution’ plugin which transforms multiple-color images into separated single color channels. Scale bar, 10 μm. f and h Average NF1 particle size in αCaMKII⁺ neurons or vGAT⁺ neurons from #20399 or #17490. Data were collected from 99 αCaMKII⁺ cells and 92 vGAT⁺ cells in #20399; 142 αCaMKII⁺ cells and 98 vGAT⁺ cells in #17490. Data is expressed as means ± SEM. Unpaired t-test, *p < 0.01, ***p < 0.001