Irdye 680 rd donkey anti mouse igg h l

The in-cell/on-cell protocol was identical for cells and decellularised matrix. Samples were washed 1 × with PBS and then fixed overnight in Histochoice Tissue Fixative (VWRVH102, VWR, Radnor, PA, USA). For in-cell western blot, the cells were permeabilised using a 0.5 % triton X-100 for 15 min. For on-cell western blot, this step was conducted with 1 × PBS. Samples were then incubated in a LI-COR blocking buffer for 1 h at 37 °C. After blocking, cells were incubated overnight at 4 °C in a primary antibody mixed in LI-COR blocking buffer. Plates were immunolabelled using antibodies raised against either the NG2/CSPG4 intracellular domain (1:200, GGQPDPELLQFCRTPNPALRNGQYWV, UIC Protein Core, Chicago, IL, USA) or the NG2/CSPG4 ectodomain (1:200, AB5320, Sigma-Millipore). Samples were washed for 15 min in 0.5 % Triton X-100 at room temperature. All samples were fluorescently tagged with secondary antibodies (1:1000; IRDye 800CW Donkey anti-Rabbit IgG (H + L) or IRDye 680RD Donkey anti-Mouse IgG (H + L), LI-COR), washed 1 × in PBS, and imaged using LI-COR fluorescence quantitative western blot. Fluorescence values were collected from a region of interest containing only the cells. Raw fluorescence values were normalised to the untreated controls. All quantified data represents four biological replicates.

On the day following the final day of behavioral testing, a subset of mice from Experiment 1 (n=23 HAP; 15 LAP3) were euthanized by cervical dislocation and brains were rapidly extracted. Bilateral punches of the dorsal striatum were extracted from a single 2mm coronal slice and rapidly frozen using liquid nitrogen. Western Blots were run to identify levels of CBi receptors following the method of Kasten et al. (2017) (link). Briefly, the tissue samples were homogenized in RIP A buffer with protease inhibitor (1ml of RIP A buffer containing lOOul of 10X PI and lOul of 0.1M PMSF) (Thermo Fisher). Sample load was calculated using a Bio-Rad Protein Assay kit. Primary antibody (Anti-Cannabinoid Receptor 1, Rabbit polyclonal to Cannabinoid Receptor 1, Abeam) was added to the PBS buffer (5% nonfat milk in 1x PBS with 0.1% Tween 20) and a secondary antibody was added at a 1:5000 dilution (IRDye 800 CW Goat anti-Rabbit IgG (H+L), LI-COR). The image was then scanned from membrane with a CLx Odyssey scanner. B-actin was used as the reference (primary β-actin mouse monoclonal antibody, secondary antibody IRDye 680RD Donkey anti-Mouse IgG (H+L), LI-COR).

Detyrosinated tubulin; rabbit polyclonal (Abcam ab48389); western blot: 1: 1,000.
Alpha tubulin; mouse monoclonal, clone DM1A (Cell Signaling #3873); western blot: 1:1,000
TTL; rabbit polyclonal (Proteintech 13618–1-AP); western blot: 1:500.
GAPDH; mouse monoclonal (VWR GenScript A01622–40); western blot: 1:1,000.
IRDye 800CW Donkey anti-Mouse IgG (H + L) (LI-COR 925–32212); western blot: 1:10,000.
IRDye 680RD Donkey anti-Rabbit IgG (H + L) (LI-COR 925–68073); western blot: 1:10,000.
IRDye 680RD Donkey anti-Mouse IgG (H + L) (LI-COR 926–68072); western blot: 1:10,000.
IRDye 800CW Donkey anti-Rabbit IgG (H + L) (LI-COR 926–32213); western blot: 1:10,000.
VASH1; rabbit polyclonal (Abcam ab199732); western blot 1:5000–1:1000
A novel antibody for human and mouse VASH1 (named anti-VASH1 Gre) was produced in rabbits by using a peptide C-RIRGATDLPKIPIPSVPTFQPTTPV-NH2 (corresponding to exposed region of the protein, see Wang, Bosc and Choi et al.)^{16 (link)} linked at the N-terminal to the keyhole limpet hemocyanin protein via the cysteine. Sera from rabbits were purified on the respective peptides. Validation of these antibodies was performed in HEK293T cells protein extracts (see Online Figure IIIA). HEK293T cultures and transfections, as well as SDS-PAGE and immunoblots, were performed as in Aillaud et al.^{11 (link)}