Guinea pig anti vgat

Neuronal cultures were fixed with 4 % paraformaldehyde and 4 % sucrose. The following antibodies were used: rabbit anti-CB1 (1:500; Synaptic System, Goettingen, Germany), guinea pig anti-vGLUT1 (1:1000; Synaptic System), and guinea pig anti -VGAT (1:750; Synaptic System). Secondary antibodies were conjugated with Alexa-488 and Alexa-555 fluorophores (Invitrogen).
Images were acquired by using a Leica Spe confocal microscope equipped with an ACS APO 63.0X1.3 objective (Leica, Wetzlar, Germany).
Pixel size was 94.8 nm_94.8 nm, and acquisition parameters (i.e., laser power, gain, and offset) were kept constant across different experimental settings. The minimum puncta size was set at three pixels.
Colocalization of two selected markers was measured by using the boolean function “and” for the selected channels. The resulting image was binarized, inverted, and used as a colocalization mask to be subtracted from single channel. The number of puncta resulting from colocalization mask subtraction (colocalizing puncta) was measured for each marker. A colocalization ratio was set as colocalized area/total puncta aerea. Fluorescence image processing and analyses were performed with the ImageJ Software (National Institutes of Health, Bethesda, MD, USA).

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.

All primary antibodies were incubated at 4°C overnight. In cases where rAAV‐mediated expression of GFP was restricted to 7 days, the GFP signal was amplified using an anti‐GFP antibody (A11122; Life Technologies) followed by a Goat Anti‐Rabbit‐488 secondary antibody (Life Technologies; both 1:500). In order to characterizes contacts onto interneurons, we stained with Rabbit anti‐vGlut1 (1:500; Synaptic Systems, #135303), Rabbit anti‐vGlut1/2 (1:500; Synaptic Systems, #135503), Guinea pig anti‐vGAT (1:1,000; Synaptic Systems, #131308), rabbit anti‐Synapsin (1:500; Millipore), and mouse anti‐Basson (1:200; ENZO) antibodies. In order to label moto neurons, we co‐stained with rabbit anti‐ChAT antibody (1:100; Abcam, #ab178850) and NeuroTrace435 (1:200; ThermoFisher, #N21479). Primary antibodies were diluted in 0.3% Triton 20 and PBS. All secondary antibodies were kept for 2 h at room temperature with gentle shaking. Secondary antibodies used are as follows: anti‐rabbit AF647 (1:500; ThermoFisher, #A32795), anti‐guinea pig Cy3 (1:500; Jackson ImmunoResearch, #706‐165‐148), anti‐guinea pig AF633 (1:500; Sigma Aldrich, #SAB4600129).