*2.11. Combined In Situ Hybridization and Immunohistochemistry*

After the detection of the FISH chromogenic reaction, sections were well washed in DEPC/PBS and incubated at RT for 1 h with blocking serum (normal goat serum 1:5 in PBS containing 0.1% Triton X-100, Sigma) and subsequently with primary antiserum ON at 4 ◦C. Primary antisera employed were: rabbit polyclonal anti-S100 (1:200, Agilent Dako, Santa Clara, CA, USA, Ref. Z 0311); mouse IgG2b, biotin-XX conjugate anti-HuC/D (1:50, Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA, Ref. A21272); mouse monoclonal anti-MAP-2 (1:50, Santa Cruz Biotechnology, Ref. Sc-74422, Dallas, TX, USA), rabbit polyclonal anti-Parvalbumin (1:100 Anti-Parvalbumin Rabbit pAB PC255L-100UL, EMD Millipore, Burlington, MA, USA). DEPC/PBS washes preceded the incubation with the secondary antibodies: goat anti-rabbit IgG (H+L) Alexa fluor™ Plus 488 (1:1000, Invitrogen by Thermo Fisher Scientific, Ref. A32731, Carlsbad, CA, USA) for anti-S100β, anti-Parvalbumin; Alexa Fluor® 488 Streptavidin conjugated (1:800, Jackson Immuno Research Labs, West Grove, PA, USA, Ref. 016540084) for HuC/D; goat anti-mouse IgG (H+L) Alexa fluor™ Plus 488 (1:1000, Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA, Ref. A32723) for MAP-2.

#### *2.12. Microscopy*

FISH images were analyzed with a Zeiss AxioScope AX 1.0 microscope (Carl Zeiss, Jena, Germany) with AxioCam MC5 and AxioVision software. Combined FISH/Immunohistochemistry images were analyzed by Leica–DM6B (Leica, Wetzlar, Germany) and processed with LasX software. The digital raw images were optimized for image resolution, contrast, evenness of illumination, and background using Adobe Photoshop CC 2018 (Adobe Systems, San Jose, CA, USA). Anatomical structures were identified according to the adult *N. furzeri* brain atlas [31].
