Rabbit anti vip

Animals were transcardially perfused with saline and 4% formaldehyde. The brains were removed, cryoprotected in 30% sucrose, and cut into 30 µm coronal sections on a frozen sliding microtome (Physitemp Instruments). Floating sections were blocked for 1 hr at room temperature in Tris buffered saline (TBS) containing 10% normal goat serum and 1% Tween-100, then incubated overnight at 4°C using in the same solution with the following antibodies: chicken anti-GFP, 1:1000 (Aves); rabbit anti-SST, 1:300 (Swant); and rabbit anti-VIP, 1:1000 (Immunostar). The sections were then washed in TBS with 1% Tween-100 three times for 15 min, incubated for 1 hr at room temperature in blocking solution with 1:1000 each of Alexa Fluor 488 goat anti-chicken and Alexa Fluor 568 goat anti-rabbit (Life Technologies). The sections were washed in phosphate buffered saline three times for 10 min, mounted on glass slides, dried, and covered with coverslips. Images of the visual cortex were captured using a Zeiss Axiovert-200 micrscope and AxioCam Mrm (Zeiss). Contrast was adjusted in ImageJ.

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.

The following primary antibodies were used: rabbit anti-cleaved caspase-3 (1:500, Cell Signaling Technologies), rabbit polyclonal anti-Calretinin (1:1000, Abcam #ab702, Cambridge, MA), mouse anti-Reelin (1:1000, Millipore #MAB5366, Burlington, MA), rabbit anti-CCK (1:1000, Frontier Institute Co. Ltd #Af350, Hakaido, Japan), rabbit anti-VIP (1:1000, ImmunoStar #20077, Hudson, Wisconsin). Fluorescent Alexa 488-conjugated antibodies (Alexa Fluor-488-conjugated goat anti-mouse IgG, Alexa Fluor-488-conjugated goat anti-rabbit IgG (1:1000, Life Technologies, Carlsbad, CA) were used as secondary antibodies.