Vibratome sections (100 μm thick) from virus-injected animals were immunolabeled with antibodies against mCherry, which was expressed from the virus, as well as antibodies against the mossy fiber marker, ZnT3. CellProfiler (Version 2.1.0) software was used to quantify the degree of overlap between mCherry and ZnT3. The average amount of overlap was 70%.
Primary antibodies: rat BrdU (AbD Serotec, MCA2060GA, 1:500), rabbit GFP (Invitrogen, A11122, 1:300), rabbit Complexin-1 (Proteintech, 10246–2–AP, 1:300), mouse GFAP (G6171, 1:500), mouse Nsf-1 (MA1–12435, 1:300) and rabbit Syn3 (OSS00018W, 1:300) from Thermo Scientific, guinea pig Iba1 (234 004, 1:500), mouse Syt1 (105 011, 1:300), guinea pig ZnT3 (197 004, 1:1000) from Synaptic Systems.
Secondary antibodies. Antibodies (all obtained from Jackson Laboratory, donkey anti-guinea pig IgG (H+L) ML, #706-225-148 and 706-175-148; donkey anti-mouse IgG (H+L) ML, #715-225-151, 715-165-151, and 715-175-151; donkey anti-rabbit IgG (H+L) ML, #711-225-152, 711-165-152, and 711-175-152 were diluted 1:400. Confocal scans were carried out using a LSM 510 Imager Z.1 microscope (Zeiss) using Plan-Apochromat 63X/1.4 and EC Plan-Neofluar 40X/1.30 oil immersion objective lenses and four excitation laser lines. Tile scans were performed using a LSM 710 confocal microscope (Zeiss) with a Plan-Apochromat 20X/0.8 objective.
Primary antibodies: rat BrdU (AbD Serotec, MCA2060GA, 1:500), rabbit GFP (Invitrogen, A11122, 1:300), rabbit Complexin-1 (Proteintech, 10246–2–AP, 1:300), mouse GFAP (G6171, 1:500), mouse Nsf-1 (MA1–12435, 1:300) and rabbit Syn3 (OSS00018W, 1:300) from Thermo Scientific, guinea pig Iba1 (234 004, 1:500), mouse Syt1 (105 011, 1:300), guinea pig ZnT3 (197 004, 1:1000) from Synaptic Systems.
Secondary antibodies. Antibodies (all obtained from Jackson Laboratory, donkey anti-guinea pig IgG (H+L) ML, #706-225-148 and 706-175-148; donkey anti-mouse IgG (H+L) ML, #715-225-151, 715-165-151, and 715-175-151; donkey anti-rabbit IgG (H+L) ML, #711-225-152, 711-165-152, and 711-175-152 were diluted 1:400. Confocal scans were carried out using a LSM 510 Imager Z.1 microscope (Zeiss) using Plan-Apochromat 63X/1.4 and EC Plan-Neofluar 40X/1.30 oil immersion objective lenses and four excitation laser lines. Tile scans were performed using a LSM 710 confocal microscope (Zeiss) with a Plan-Apochromat 20X/0.8 objective.