Alexa fluor 488 555 594

Whole-mount preparations were placed under a microscope with fluorescent illumination (Olympus BX51WI; light source AMH-200-F6S; Andor Technology-Oxford Instruments). Micropipettes filled with dextran-coupled fluorophores (see above; 3 mM in frog ringer, Alexa Fluor 488/555/594 and cascade blue dextran, 10 kD, Thermo Scientific) were mounted to the head stage of a single cell electroporator. We lowered the pipette tip toward the OB surface using a micromanipulator before penetrating it into the mitral cell layer and applying a voltage pulse train (50 V, 300 μs pulse length, 500 ms train duration, 300 Hz; details see.⁶⁸ (link) We used prior bulk electroporation with fluorophore-coupled dextrans, WGA application, or the Katushka fluorescence of the tubb2b transgenic reporter line³⁰ (link) to identify the mitral cell layer.

Immunocytochemistry was performed as described previously with some modifications^{18 (link)}. Cells were fixed with 4% paraformaldehyde/PBS and permeabilized with 0.5% Triton X-100/PBS for 15 min. Then, cells were blocked with 5% foetal bovine serum/PBS for 30 min. For primary antibodies, cells were incubated at 4 °C for 16 h with the respective antibodies, such as anti-SOX1 (1:100, R&D Systems), anti-SOX2 (1:100, R&D Systems), anti-PAX6 (1:100, Stemgent, San Diego, CA, USA), anti-Nestin (1:200, eBioscience, San Diego, CA, USA), anti-TUJ1 (1:1,500, Covance, Berkeley, CA, USA), anti-MAP2 (1:1,500, Millipore), anti-TBR1 (1:200, Abcam, Cambridge, UK), anti-GFAP (1:1,500, DakoCytomation, Carpinteria, CA, USA), anti-p62 (1:100, Abcam), anti-NeuN (1:100, Millipore), anti-cleaved caspase 3 (1:800, Cell Signaling Technology, Beverly, MA, USA), anti-GRP78/BIP (Abcam), or anti-GADD153/CHOP (Santa Cruz Biotechnology). Cells were washed with PBS and then incubated for 60 min with secondary antibodies such as Alexa Fluor 488, 555, 594, or 647 (Thermo Fisher Scientific). Nuclei were counterstained with Hoechst 33342 (Dojindo, Kumamoto, Japan). The cytoplasm was stained with HCS CellMask Deep Red Stain (Thermo Fisher Scientific).