Neurotrace fluorescent nissl stain

For dual staining of calbindin and NeuroTrace, 10 µm or 40 µm tissue sections were immunolabeled with primary antibody as described above (calbindin; 1:10,000; CD38). We then added NeuroTrace fluorescent Nissl stain (Molecular Probes, Eugene, OR, USA), diluted to 1:1000, directly to the secondary antibody cocktail. Alexa-Fluor^®-conjugated secondary antibodies (1:1500; Life Technologies; A31570) were used to amplify and detect the calbindin signal. After three PBS rinses, we mounted the stained tissue sections with FLUORO-GEL mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA) onto glass slides before imaging.

Mice were dispatched using 750–1000 mg kg⁻¹ avertin and transcardially perfused with 1× PBS solution, followed by 4% paraformaldehyde (PFA). Brains were extracted and post fixed in 4% PFA at 4 °C for 24 h. Brains were transferred to 1× PBS and 50 µm coronal slices were prepared using a vibratome. For immunostaining, each slice was placed in PBS + 0.3% Triton X-100 (PBS-T), with 5% normal goat serum for 1 h and then incubated with primary antibody at 4 °C for 24 h. Slices then underwent three wash steps for 10 min each in PBS, followed by a 2 h incubation with secondary antibody at room temperature. After three more wash steps of 10 min each in PBS-T, slices were mounted using VECTASHIELD mounting medium on positively charged glass slides. Antibodies used for staining were as follows: chicken anti-GFP (1:1000 dilution, Life Technologies) and anti-chicken Alexa-488 (1:500 dilution), rabbit anti-RFP (1:1000 dilution, Rockland Inc.) and anti-rabbit Alexa-555 (1:500 dilution), cFos was stained with rabbit anti-cFos (1:400 dilution, Santa Cruz) and anti-rabbit Alexa-555 (1:500 dilution), and nuclei were stained with DAPI (1:3000 dilution, Sigma). For counterstaining in Fig. 5, brain sections were incubated with Neuro Trace Fluorescent Nissl Stain (1:100 dilution, Molecular Probes) in 1× PBS for 1 h after secondary antibody staining.