Alexa fluor 594 conjugated secondary antibody

Culture media and supplements, collagenase type XI, histopaque-1077, DMSO, EDTA, IBMX, carbachol, clonidine, LiCl, exendin-4, forskolin, agarose, bionic buffer and BSA were obtained from Sigma-Aldrich (Dorset, UK). DNeasy Blood and Tissue, RNeasy Mini and QuantiTect SYBR Green PCR kits and qPCR primers for mouse and human CB₁ (CNR1), GPR119, GPR18, GPR92 (LPAR5), delta-opioid receptor (OPRD1), transient receptor potential cation channel subfamily V member 1 (TRPV1), GPR3, GPR6, GPR12, and ACTB were from Qiagen (Manchester, UK). PCR primers for Gpr55 genotyping were from Eurofins Genomics (Wolverhampton, UK). SR141716A was from Tocris Bioscience (Abingdon, UK). AM251 and rabbit anti-Ki67 primary antibody were from Abcam (Cambridge, UK). cAMP HiRange and IP-one (IP₁) assays were from Cisbio (Codolet, France). TaqMan RT-PCR kit, 100 base pairs (bp) DNA ladder, SYBR® DNA gel stain, HEPES, HBSS and DAPI were from Thermo Fisher Scientific (Paisley, UK). Caspase-Glo 3/7 and GoTaq^® G2 Green Master Mix were from Promega (Southampton, UK). Recombinant TNFα, IFNγ and IL-1β were from PeproTech EC (London, UK). Guinea pig anti-insulin was obtained from Dako (Cambridge, UK). AlexaFluor 488- and AlexaFluor 594-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (Newmarket, UK).

IHC was carried out as previously described (Xu et al., 2014 (link)) using the following antibodies: anti-L1 (MAB5272, MilliporeSigma), anti-ROBO1 (NBP2-20195, Novus Biologicals), anti-ROBO2 (NBP1-81399, Novus Biologicals), anti-ROBO3 (Sabatier et al., 2004 (link)), anti-TAG1 (4D7, Developmental Studies Hybridoma Bank), and Alexa Fluor 594-conjugated secondary antibodies (Jackson ImmunoResearch). The antibodies were used at a final concentration of 0.5–1 μg/ml.

NP cells were plated in a six-well plate. After treatment, NP cells were washed three times with PBS, fixed with 4% paraformaldehyde, and then infiltrated with 0.1% Triton X-100 in PBS. After blocking the cells with 5% bovine serum albumin, they were incubated with mouse monoclonal anti-LC3B (1:1000 dilution, #83506, CST). Finally, the cells were incubated with Alexa Fluor 594-conjugated secondary antibodies (715–585–150; Jackson ImmunoResearch) and stained with the nuclear staining dye DAPI. A Zeiss LSM800 confocal microscope was used to observe the stained cells in five random different fields for each slide.

Primary astrocytes were seeded onto Poly-D-Lysine (PDL)-coated coverslips and maintained in a humidified incubator for 2 days. Thereafter, astrocytes were transfected with the indicated CRISPR/Cas9 plasmids by electroporation and incubated for 48 h. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100. After blocking for 1 h in 0.1% Triton X-100 and 3% BSA, the cells were incubated with mouse anti-TREK-1 (Santa Cruz Biotechnology, Dallas, TX, USA; RRID: sc-398449, 1:100) or mouse anti-TWIK-1 (Santa Cruz Biotechnology, Dallas, TX, USA; RRID: sc-517040, 1:100) antibodies at 4 °C overnight. After washing with PBS, cells were incubated with Alexa Fluor 594-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA, 1:400) for 1 h at room temperature. After three rinses with PBS and staining with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei, images were acquired by confocal microscopy using a Nikon A1 confocal microscope (Nikon Instruments Inc., Melville, NY, USA).