Shandon permafluor mounting medium

Mice at 6 to 10 months were anesthetised with 0.3 mg/g Pentobarbital (Mebunat 60 mg/mL) mixed 50:50 with 0.9% NaCl. Mice were perfused transcardially on ice bedding using 10–20 mL 0.9% NaCl followed by 25–50 mL of buffer comprising 4% paraformaldehyde and 0.125% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer (NaH₂PO₄-Na₂HPO₄, pH 7.2). The brain was post-fixed in 50 mL 4% paraformaldehyde in phosphate buffer for 4–12 h at +4 °C. Coronal sections of 180 to 200 µm were sectioned using a vibratome. The nuclei were visualized using DAPI (4′,6-diamino-2-phenylindole, dihydrochloride; InvitrogenSelected)). Neurons were injected by iontophoresis with Lucifer yellow dye (Invitrogen) using pulled borosilicate glass tubes (World Precision Instruments). The DC current source was 2–6 nA from a dual micro-iontophoresis current generator, model 260 (World Precision Instruments). After dye loading, brain slices were transferred to a slide and mounted using Shandon PermaFluor mounting medium (ThermoFisher).

Mice at 6–8 months were anesthetized with 0.3 mg/g Pentobarbital (Mebunat 60 mg/ml) mixed 50:50 with 0.9% NaCl. Mice were perfused transcardially on ice bedding using 10–20 ml 0.9% NaCl followed by 25–50 ml of buffer comprising 4% paraformaldehyde and 0.125% glutaraldehyde in 0.1 M Sorensen’s phosphate buffer (NaH₂PO₄–Na₂HPO₄, pH 7.2). The brain was post-fixed in 50 ml 4.0% PFA in phosphate buffer for 4–12 h at +4°C. Coronal sections of 180–200 μm were sectioned using a vibratome. The nuclei were visualized using DAPI (4^′,6-diamino-2-phenylindole, dihydrochloride (Invitrogen). Pyramidal neurons in the motor cortex and the hippocampus were located according to the Atlas of C57BL/6 mouse brains (Hof et al., 2000 ). Selected neurons were injected by iontophoresis with lucifer yellow dye (Invitrogen) using pulled borosilicate glass tubes (World Precision Instruments). The DC current source was 2–6 nA from a dual micro-iontophoresis current generator, model 260 (World Precision Instruments). After dye loading, brain slices were transferred to a slide and mounted using Shandon PermaFluor mounting medium (ThermoFisher).