Cfi super plan apo 20 objective

Two male CaMKIIα/GFP-FVB/AntF1 mice (P53 and P67; Wang et al., 2013 (link)) were anesthetized and transcardially perfused with 4% PFA in 0.1 M PB, then the brains were removed and cut into 50-μm sections with a vibratome (Leica VT1000S). Immunostainings detailed in Table 1 were performed by incubating the slices in the primary antibody mixture with 2% Triton X-100, 2% NDS and 0.05% Na-azide in 0.1 M PB for 24 h at RT and 2 d at 4°C, then after thorough washing with 0.1 m PB, sections were incubated in the secondary antibody mixture (all 1:500, from The Jackson Laboratory) in 0.1 M PB for 4 h at RT. After washing, sections were mounted in Vectashield (Vector Laboratories) and 3D images were obtained using a Nikon C2 confocal microscope with Nikon CFI Super Plan Apo 20× objective (N.A. 0.75; z step size: 2 μm, xy: 0.31 μm/pixel). Colocalization of the neurochemical markers in the neurons was examined and quantified manually with the Neurolucida 10.53 software (MBF Bioscience).

After in vitro recordings slices were fixed in 4% paraformaldehyde overnight at 4°C and biocytin content of the recorded neurons was visualized using Alexa 647-conjugated streptavidin (1:10,000 or 1:20,000, Invitrogen). mCherry and GFP or EYFP signals in the neurons were enhanced by immunostainings using the following antibodies: rat anti-RFP (1:1000, Chromotek) revealed by Cy3-conjugated donkey anti-rat secondary antibody (1:500, The Jackson Laboratory) and chicken anti-GFP (1:1000, SYSY) revealed by Alexa 488-conjugated donkey anti-chicken antibody (1:500, The Jackson Laboratory). Slices were mounted in Vectashield (Vector Laboratories) and 3D images were obtained using a Nikon C2 confocal microscope with Nikon CFI Super Plan Apo 20× objective (N.A. 0.75; z step size: 1 μm, xy: 0.31 μm/pixel). Images were processed and analyzed with the NIS Elements AR 5.30.01 software (Nikon Instruments).