Axioskop 40 microscope

The heads of E14.5 fetuses were harvested as described above and fixed in formaldehyde for 48 hours, then embedded in paraffin and cut in 5 μm coronal or rostro-caudal sections. In situ hybridization of Dio3 mRNA was performed in selected sections of 2 animals per genotype utilizing the RNAscope technique (Advanced Cell Diagnostics, Bio-Techne) following the manufacturer’s suggested procedures. We used the RNAscope Mm-Dio3 probe (catalog number 561641) and the ACD 2.5 HD Detection kit (RED). As a technical negative control, we use the bacterial probe DapB supplied by the manufacturer, while experimental negative controls included tissue sections from mice lacking the full Dio3 gene (Dio3^DEL/DEL mice). Some tissue sections were counterstained with hematoxylin and mounted with EcoMount (catalog EM897L, Biocare Medical), while other sections were mounted with DAPI Fluoromount-G (catalog 0100-20, Southern Biotech). Bright-field or fluorescent images of the mRNA signal were taken, respectively, with a Zeiss Axioskop 40 microscope or a Leica SP8 confocal microscope utilizing LAS X software. For anatomic reference, adjacent tissue sections were stained with H&E at our Histology Core facility following standard procedures.

The heads of E18.5 fetuses were harvested as described above and fixed in 4% formaldehyde for 48 h. After fixation they were paraffin-embedded and cut in five microns coronal or rostro-caudal sections. In situ hybridization of Klf9 and Nrgn mRNAs was performed in selected sections of two animals per genotype utilizing the RNAscope technique (Advanced Cell Diagnostics, BioTechne Corporation, Newark, CA, United States) following the manufacturer’s suggested procedures. We used the RNAscope Mm-Klf9 and Mm-Nrgn probes (catalog numbers 488371 and 499441, respectively) and the ACD 2.5HD Detection kit (RED). As a negative control, we used the bacterial probe DapB supplied by the manufacturer. Some tissue sections were counterstained with hematoxyline and mounted with EcoMount (catalog # EM897L, Biocare Medical, Pacheco, CA, United States), while other sections were mounted with DAPI Fluoromount-G (Catalog # 0100-20, Southern Biotech, Birmingham, AL, United States). Bright field or fluorescent images of the mRNA signal were taken, respectively, with a Zeiss Axioskop 40 microscope or a Leica SP8 confocal microscope utilizing LAS X software. For anatomic reference, adjacent tissue sections were stained with H&E at our Histology Core facility following standard procedures.