Plan apo vc 20 objective

Two offspring of VIP-IRES-cre mice crossed with Ai14 reporter mice were transcardially perfused with 4% PFA in 0.1 m PB. The brain was removed from the skull and resectioned into 50-μm-thick horizontal slices. To estimate the GABAergic inputs received by tdTomato-expressing VIP+ INs, sections containing the BLA were processed for immunostaining with the following antibodies: guinea pig anti-VGAT (Synaptic Systems; 1:1000), rat anti-RFP (Chromotek; 1:1000), and rabbit anti-CB1 (Cayman Chemicals; 1:1000). VGAT was visualized with Alexa Fluor 488-conjugated donkey anti-guinea pig (Jackson ImmunoResearch; 1:500), tdTomato signal was enhanced by Cy3-conjugated donkey anti-rat (Jackson ImmunoResearch; 1:500), and CB1 was visualized with Cy5-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch; 1:500). Confocal images were obtained using a Nikon C2 confocal microscope (Plan Apo VC 20× objective; numerical aperture, 0.75; z-step size, 1 μm; xy, 0.62 μm/pixel). The image analysis was performed using Neurolucida Explorer.

Ca²⁺ measurements were performed essentially as described previously [20 (link), 78 (link), 79 (link)]. SH-SY5Y cells were loaded with 2 μM Fluo4-AM and/or Rhod2-AM dye (Invitrogen) in external solution (145 mM NaCl, 2 mM KCl, 5 mM NaHCO₃, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM Na-HEPES, pH 7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at 37 °C, followed by washing in external solution for 15 min at 37 °C. Fluo4 and Rhod2 fluorescence were timelapse recorded (1 s intervals) at 37 °C using either an Axiovert S100 microscope (Zeiss) driven by MetaMorph (Molecular Dynamics) and equipped with GFP (Fluo4) and DsRed (Rhod2) filtersets (Chroma Technology), a 40× Plan-Neofluar 1.3NA objective (Zeiss), and a Photometrics Cascade-II 512B36 EMCCD camera or a Nikon Ti-E microscope using a CFI Plan Apo VC 20× objective and Nikon Andor Neo sCMOD high-resolution camera and appropriate filter sets. The cells were kept under constant perfusion with external solution (0.5 ml/min). Inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca²⁺ release from ER stores was triggered by application of 100 μM Oxotremorine-M for 2 min. Ca²⁺ levels were calculated as relative Rhod2 or Fluo4 fluorescence compared to baseline fluorescence (F/F₀) at the start of the measurement. Oxotremorine-M was from Santa Cruz Biotechnology and was dissolved in water.

Some slices prepared from AAV-injected VIP-IRES-cre::GAD65-EGFP mice were fixed in 4% PFA in 0.1 m PB and after overnight incubation were resectioned at a 40 μm thickness. Different sections were processed for single immunostaining with rabbit anti-pro-CCK (Frontiers Institute; 1:1000), guinea pig anti-NPY (Synaptic Systems; 1:1000), or rabbit anti-CaMKII (Abcam; 1:250). Visualization was performed with Cy5-conjugated secondary antibodies. In addition, double immunostaining was obtained using a mixture of rat anti-SOM (Millipore; 1:500) and guinea pig anti-PV (Synaptic Systems; 1:1000) antibodies or a mixture of rabbit anti-VIP (Immunostar; 1:4000) and guinea pig anti-CR (Synaptic Systems; 1:1000) antibodies. Here, DyL405-conjugated and Cy5-conjugated appropriate secondary antibodies were used to visualize the antigen–antibody complexes. Confocal images were obtained with a Nikon C2 confocal microscope (Plan Apo VC 20× objective; numerical aperture, 0.75; z-step size, 2 μm; xy, 0.62 μm/pixel) and the analysis was conducted using Neurolucida Explorer (RRID:SCR_001775). Analysis of the colocalization between CCK and EGFP was limited to neurons with large soma, as these are known to be CB1+ basket cells (Szabó et al., 2014 (link)) and we did not find overlap between CCK and VIP in these cells in this mouse line. In addition, data for VIP+ and CR+ INs were combined.