Cy3 conjugated donkey anti rat

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.

Third-instar wandering larvae were dissected in PBS and fixed in 4% formaldehyde in PBS. Samples were washed three times after fixation with PBS containing 0.3% Triton X-100 and transferred to blocking solution (PBS containing 5% normal donkey serum and 0.3% Triton X-100). Specimens were incubated overnight at 4 °C with primary antibodies diluted in blocking solution. Primary antibodies were washed four times with PBS containing 0.3% Triton X-100 before the incubation overnight at 4 °C with secondary antibodies. Then, the samples were washed four times with PBS containing 0.3% Triton X-100. The primary antibodies used were as follows: rabbit anti-PatJ (1:1000), rat anti-Dpn (Abcam, 1:50), and guinea pig anti-L’sc (1:1200). The following secondary antibodies (Jackson) were used at 1:200 dilutions: FITC-conjugated donkey anti-rabbit, Cy3-conjugated donkey anti-rat, and Alexa Fluor 647-conjugated donkey anti-guinea pig. Specimens were mounted with VectaShield mounting media (Vector) and viewed on a Zeiss LSM880 confocal microscope. ZEN software (Zeiss) was used for preparing three-dimensional images.

Paraffin sections (6 μm) were deparaffinized, rehydrated, prepared, and double-stained with the following antibodies: (i) rat anti-CD4 (1:500; BD Pharmingen, California, USA) with rabbit anti-IL-17 (1:500; Novus Biologicals, Littleton, Colorado, USA) and (ii) rat anti-CD4 (1:500; BD Pharmingen, California, USA) with goat anti IL-10 (1:200; R&D, California, USA). The sections were incubated with the antibodies in a humidified chamber overnight at 4 °C. After being washed with PBS, the sections were incubated with a mixture of Alexa 488-conjugated donkey anti-goat (1:500; Jackson ImmunoResearch, Pennsylvania, USA) and Cy3-conjugated donkey anti-rat (1:500; Jackson ImmunoResearch, Pennsylvania, USA) secondary antibodies for 2 h at room temperature. All of the sections were counterstained with DAPI (1:10 000; #PP0131, Beyotime Biotechnology, Shanghai, China). Images were obtained using a FluoView confocal microscope (#FV3000, Olympus, Tokyo, Japan). Immunostaining quantification was completed using ImageJ software.