Goat anti guinea pig af488

NIH cells (Fig. 2) were seeded onto cover slips placed in 24-well plates. After 24 h, the medium was changed to new medium containing 360 μM oleic acid. After 20 h, the cells were fixed with 2% formaldehyde for 10 min and stored at 4°C in PBS. For immunolabeling, the cells were treated with Avidin/Biotin Blocking kit (Vector Laboratories), and 1% BSA in PBS for 20 min. The cells were then incubated 2h at room temperature with mouse anti-vinculin (1:100, clone SPM227, Abcam), followed by 30 min biotinylated monovalent donkey anti-mouse IgG (1:200, Jackson ImmunoResearch) and 30 min with Atto425-conjugated streptavidin (1:200, ATTO-TEC). The cells were then incubated for 2h at room temperature with guinea pig anti-ADRP (1:400, Fitzgerald), rabbit anti-mouse syntaxin 5 (1:150, Synaptic System) and rat anti-mouse LAMP1 (1:100, clone 1D4B, Abcam) followed by 30 min incubation with goat anti-guinea pig AF488 (1:400, Life Technologies), goat anti-rabbit Cy3 (1:250, Jackson ImmunoResearch), mouse anti-rat PerCP-eFluor710 (1:100, Affymetrix) and phalloidin-Atto594 (5 μL/mL, Sigma-Aldrich); in the last 5 min, 3 drops of DAPI (2 μg/mL) was added to each well. All incubation steps contained 0.1% saponin and all washing steps contained 0.05% saponin for permeabilization. The cover slips were mounted onto microscope slides with Prolong Gold antifade reagent (Life Technologies).

Plated cells were fixed in 4% paraformaldehyde (PFA; Fisher Scientific) for 20 minutes at room temperature and rinsed with PBS. Nonspecific labeling was blocked and the cells permeabilized with 5% normal goat serum (Millipore) and/or 5% normal donkey serum (Millipore) and 0.2% Triton X-100 (Sigma) in PBS for 30 minutes at room temperature. Cells were rinsed with PBS and then incubated with primary antibodies for one hour at room temperature or overnight at 4°C. Cells were subsequently labeled with the appropriate fluorescently-tagged secondary antibodies. Hoechst nuclear dye was used to label nuclei. Primary antibodies used were rabbit anti-Peripherin (Millipore, AB1530), mouse anti-βIII Tubulin (Tuj1, Promega, G7121), rabbit anti-NTRK1 (Millipore, 06-574), rabbit anti-TRPV1 (Novus Biologicals, NBP1-97417), mouse anti-GFAP (Cell Signaling, 3670), rabbit anti-Parvalbumin (Calbiochem, PC255L), mouse anti SMI32R (Covance, SMI-32R), guinea pig anti-VGlut1 (Millipore, AB5905), and myelin protein zero (ProteinTech, 10572-1-AP). Secondary antibodies included donkey anti-mouse AF488 (Invitrogen, A21202), goat anti-rabbit RhoRed (Invitrogen, R6394), and goat anti-guinea pig AF488 (Life Technologies, A11073).

DAPI (4′, 6′-diamidino-2-phenlyindole, #D1306), Isolectin GS-IB₄ (#I21411, #I21412) and 10X PBS (#70011) were purchased from Invitrogen (Carlsbad, CA). Sodium borohydride (#480886), 95% paraformaldehyde (#441244), ethanol (#E7148), Triton X-100 (#T8532) and Sudan Black B (#199664) were purchased from Sigma-Aldrich (St. Louis, MO). Chicken anti-GFP (1:2000; #AB13970) was purchased from Abcam (Cambridge, MA). Rabbit polyclonal anti-dsRed (1:1000; #632496) was purchased from Clontech (Mountain View, CA). Guinea pig anti-A_2A adenosine receptor (1:4000; A_2A-GP-Af1000) was purchased from Frontier (Hokkaido, Japan). Rabbit anti-phospho-ERK (1:1000; #9101) was purchased from Cell Signaling (Beverly, MA). Rabbit anti-Iba (1:1000; #019-19741) was purchased from Wako (Osaka, Japan). AF488 goat anti-chicken (1:1000; #A11039), AF488 goat anti-guinea pig (1:1000; #A11073) and AF568 goat anti-rabbit (1:1000; #A11011) were purchased from Invitrogen.