Prb 278p

Human and Chimp organoids were infected with MOI of 0.1 and analyzed after 24 and 96 hours p.i. Organoids were cryosectioned at 20 um. Immunofluorescence was performed after blocking sections in a solution with 0.1% Triton and 3% BSA (Gemini) for 1 hour at room temperature. The primary antibodies were diluted in a solution with 0.1% Triton and 3% BSA, and the sections were incubated with following antibodies: anti-ZIKV, anti-flavivirus, anti-MAP2, anti-cleaved-caspase-3 and anti-Sox2, all mentioned above, and anti-PAX6 (rabbit, Covance PRB-278P, 1:100), anti-TBR1 (rabbit, Abcam ab31940, 1:300), CTIP2 (rat, Abcam ab18465, 1:100) and Ki67 (rabbit, Abcam ab15580, 1:100). The sections were blocked with 0.1% Triton (Sigma-Aldrich) and 3% BSA for 30 min at room temperature and the secondary antibodies previously diluted, the same mentioned above, were added. The nuclei were stained with DAPI, as mentioned above and slides were mounted with DPX (Sigma-Aldrich).

Immuno-cytochemistry of the human and monkey telencephalic organoids was started first rehydrating frozen slides in PBS. Blocking was done 1 hour RT in PBS- 10% normal donkey serum (Sigma-Aldrich, D9663) plus 0.5% tween-20. Incubation with primary antibodies was performed in PBS- 10% normal donkey serum plus 0.5% tween-20 at 4 °C, ON. The following primary antibodies were used at the concentration indicated: PAX6 (BioLegend, PRB-278P; 1:200); NKX2-1 (ABCAM, ab76013; 1:500); LMX1A (ABCAM, ab76013, 1:200); SP8 (ABCAM, ab73494; 1:200); ZIC4 (Lsbio, LS-B9905-50; 1:100); Galanin (Millipore Sigma, AB2233; 1:100); GALR2 (ABCAM, ab188753; 1:100); GALP (Novus biological, NBP2-84950; or Thermo Fisher Scientific, BS-11526R; 1:100); SOX2 (R&D Systems, AF2018; or MAB2018; 1:200); HuC/D (Thermo Fisher Scientific, A-21271; 1:500); KI67 (ABCAM, ab15580; 1:200); GABA (Sigma-Aldrich, A2052; 1:100); CTIP2 (ABCAM, ab18465; 1:1000). Secondary antibody incubation was performed in PBS 1 hour RT. Secondary antibodies were Alexa Fluor 488-, 594-, or 647-conjugated AffiniPure Donkey anti-IgG (1: 200; Jackson ImmunoResearch). Nuclei were counterstained with DAPI (Sigma, D8417). Finally, the slides were mounted using Vector mounting medium.

CS sections were washed with PBS to remove residual OCT/sucrose and then blocked in a solution consisting of PBS with 10% normal donkey serum (NDS), 0.3% Triton X-100 and 1% BSA for 1 hour at room temperature. The sections were then incubated with primary antibodies diluted in a solution consisting of PBS with 2% NDS and 0.1% Triton X-100 overnight at 4°C. The following primary antibodies were used for staining: anti-PAX6 (mouse, 1:300, BioLegend, PRB-278P), anti-CTIP2 (rat, 1:300, abcam, ab18465), anti-TBR2 (mouse, 1:100, R&D Systems, MAB6166), anti-SOX9 (goat, 1:300, R&D Systems, AF3075), anti-SSTR2 (mouse, 1:100, R&D systems, MAB4224), anti-MAP2 (guinea pig, 1:200, Synaptic Systems, 188 004), and anti-TUBB3 (rabbit, 1:200, Cell Signaling TECHNOLOGY, #5568S). Sections were washed with PBS to remove primary antibodies, and incubated with secondary antibodies (Alexa Fluor dyes, Life Technologies) diluted 1:1000 in PBS with 2% NDS and 0.1% Triton X-100 for 1 hour at room temperature. Sections were washed with PBS and nuclei were stained with Hoechst 33258 (Thermo Fisher Scientific, H3569) for 5 minutes at room temperature. Sections were mounted on glass slides with Aqua-Poly/Mount (Polysciences, Inc., 18605-5) and imaged with a Leica TCS SP8 confocal microscope. Images were processed with ImageJ (Fiji).