Goat anti sox2 antibody

SHIELD-processed and cleared organoids were stained using an adapted version of the eFLASH protocol^{27 (link),44 (link)}. We incubated organoids in eFLASH sample overnight at room temperature. Organoids were then placed in the SmartLabel system (LifeCanvas) with 1.4 mL sample buffer in the sample cup. For SCOUT pipeline, we added 6µL Syto16 (1 mM solution, ThermoFischer #S7578), 15 µg goat anti-SOX2 antibody (R&D Systems #AF2018), 10 µg Fab fragment anti-goat IgG Alexa Fluor 594 (Jackson ImmunoResearch #805-587-008), 30 µg rabbit anti-TBR1 Alexa Fluor 647 (Cell Signaling Technology #45664S). Additional antibodies used for whole organoid staining are anti-β3-tubulin (mouse monoclonal, Biolegend #657408), anti-MAP2 (mouse monoclonal, Biolegend #801803) and vimentin (rabbit monoclonal, Cell Signaling Technologies #45664). All antibodies used in this study are listed in Supplementary Table 1.

Sections (4µm) from formalin fixed, paraffin embedded cervical biopsies were dewaxed in xylene and rehydrated through alcohol to water. Antigen retrieval was performed in 0.9 M citrate buffer in a pressure cooker in an 800W microwave oven for 15 minutes at full power. After cooling, slides were blocked with avidin-biotin block followed by protein block (Insight Biotechnology Ltd, UK) and stained with goat-anti-SOX2 antibody (R&D Systems, UK) or mouse monoclonal anti-TP63 antibody (AbCam, UK) overnight at 4°C. Sections were then washed, incubated with rabbit polyclonal biotinylated anti-goat or anti-mouse IgG (Vector Labs, UK), washed, incubated with streptavidin-HRP, washed and colour developed using DAB solution (Vector Labs). Sections stained with only secondary antibody (no primary control) were included in each run. Parallel sections from 11 cases were stained with SOX2 and TP63. Tumour cells were evaluated for their nuclear expression of the transcription factors. There was no significant difference between TP63 and SOX2 levels (Wilcoxon ranked pairs test). For each section stained, five fields were photographed at ×200 magnification. Using the ImageJ software “cell counter” plug-in, each picture was overlaid with a grid and nuclear +ve and –ve tumour cells counted. At least 150 cells per field (750 per section) were counted.

Brain cryosections were incubated with a primary mouse anti-ZIKV E protein antibody (Abcam) (1:500 dilution), goat anti-SOX2 antibody (R&D) (1:500 dilution), or goat anti-Olig2 antibody (R&D) (1:50 dilution) overnight at 4°C. The sections were then incubated with Alexa Fluor 488-conjugated anti-mouse and 594-conjugated anti-goat secondary antibodies (Thermo Fisher) (1:300 dilution) for 2 h at 37°C. Finally, the sections were incubated with DAPI (4′,6-diamidino-2-phenylindole) (1:1,000 dilution) for 10 min. Images were acquired by confocal laser scanning microscopy (Zeiss).