Cy3 conjugated anti rabbit secondary antibody

In Arabidopsis, whole-mount immunolocalization was performed following the published protocol^{69 (link)}. Antibodies were diluted as follows: 1:1000 for rabbit anti-PIN1^{15 (link)} (produced and processed in lab); 1:1000 for rabbit anti-PIN2^{70 (link)} (produced and processed in lab); and 1:600 for CY3-conjugated anti-rabbit secondary antibody (Sigma, C2306). In pea, water lanolin pastes containing IAA (0.16 μM), or IAA/GR24 (0.16 µM/0.09 µM) were applied on the stem stump or on the stem 2 mm below lateral incision. Immunolocalization was performed on longitudinal pea stem segments as described for Arabidopsis stem^{69 (link)}. The Arabidopsis anti-PIN1 antibody can also recognize the homologous PIN protein in pea^{4 (link)}. Antibodies were diluted as follows: 1:1000 for rabbit anti-PIN1^{15 (link)} (produced and processed in lab); and 1:500 for CY3-conjugated anti-rabbit secondary antibody (Sigma, C2306). All the fluorescence signals were evaluated on Zeiss LSM 700, Zeiss LSM 710, Zeiss Observer. Z1, Leica TCS SP2, Olympus Fluoview FV1000, or Olympus Fluoview 200 confocal scanning microscopes. Unless otherwise noted, the same microscope settings were usually used for each independent experiment and pixel intensities were taken into account when comparing the images between different samples. Images were finally assembled in Adobe Photoshop CC 2015 and Adobe Illustrator CS6.

Log-phase promastigotes were fixed in 4% paraformaldehyde for 15 min followed by washing (in PBS) and permeabilization in 0.1% Triton X-100 (in PBS) for 15 min. The parasites were allowed to adhere on to poly-L-lysine coated slides for 1 h. The parasites were then blocked in 2% BSA (in PBS) followed by incubation with anti-LdeK1 antibody (1:200) for 1 h and cy3-conjugated anti-rabbit secondary antibody (Millipore) for 1 h. DABCO (Sigma) was used as an anti-fading agent. Images were acquired using a confocal laser scanning microscope (Ziess LSM510 Meta, Belgium).

IHC was performed to confirm ChETA expression both in cells within the injection site in the MDT and in terminals within the mPFC. After AST, rats were sacrificed via rapid decapitation and brains were postfixed in 4% paraformaldehyde. Brains were sectioned at 40 μm on a vibratome for free-floating IHC. After peroxidase inactivation, sections were incubated in primary rabbit anti-GFP antibody (1:5000; Cell Signaling, RRID:AB_10692764) followed by Cy3-conjugated anti-rabbit secondary antibody (1:1000; Millipore, RRID:AB_92489) and counterstained using DAPI. Alternate sections were used to confirm placement of electrodes and optical implants. Viral expression was confirmed if GFP label was seen in both the MDT and in mPFC terminals. It is important to note that viral spread and uptake could not be precisely localized in MDT six to seven weeks after injection, and likely included thalamic regions adjacent to the MDT. Nonetheless, any terminals labeled in the mPFC, and thus activated optically, arose from the thalamic afferent. Rats would be eliminated if GFP expression was not observed in terminals in the mPFC. 2 rats were eliminated due to these criteria. These rats were in the NS-LTD and CUS-LTP groups.