Goat anti rabbit cy3

Brains were dissected using forceps (FST 11413–11) in 1X PBS and then transferred to PBS on ice. Immunohistochemistry was performed as previously described with some modifications [61 (link)]. Brains were fixed in 1X PBS with 4% paraformaldehyde for 30 min. After washing three times (5 min each) in PBST (1X PBS with 0.5% Triton-100), antigen retrieval was performed by incubating brains in 1X PBS with 0.1% SDS and 20% H₂O₂ for 30 min. Next, brains were washed three times in PBST (5 min each), blocked in 10% NGS for one hour, and incubated in the primary antibody solution for three days. After washing overnight in PBST, brains were transferred to the secondary antibody solution overnight and then washed in PBST overnight. Samples were mounted using Vectashield Mounting Medium (VECTASHIELD) and imaged using a Nikon A1 Confocal Microscope. Primary antibodies used were rabbit anti-dsRed (1:200, Takara Bio), rabbit anti-GFP (1:500, Invitrogen), mouse anti-GFP (1:500, Sigma-Aldrich), and mouse anti-HA (1:200, Invitrogen). Secondary antibodies included goat anti-rabbit Cy3 (1:250, Jackson Immuno), goat anti-rabbit Alexa Fluor 488 (1:250, Jackson Immuno), goat anti-mouse CF488A (1:250, Sigma-Aldrich), and goat anti-mouse TRITC (1:250, Invitrogen).

Biopsies taken from fresh lung cancer resection specimens were snap frozen in liquid nitrogen and stored at −80°C until further processing. Sections of 5 μm, mounted on poly‐L‐lysine coated slides, were stored at −80°C until staining. Immunofluorescent staining of GLUT1, CAIX, MCT1, MCT4 and vascular density were as described previously by Meijer et al.17Staining for SLC1A5 and GLS2 was performed on two consecutive tumor sections by incubating the sections with rabbit anti‐SLC1A5 (Abcam, Cambridge, UK) and rabbit anti‐GLS2 (ThermoFischer Scientific, Waltham, Massachusets, USA) respectively, diluted 1:150 in PAD (Bio‐Rad Laboratories Inc., Richmond, CA, USA), overnight at 4°C for SLC1A5 and 45 minutes at 37°C for GLS2. For SLC1A5, the second and third incubation took 30 minutes at 37°C with goat anti‐rabbitCy3 (Jackson Immuno Research Laboratories Inc.; West Grove, PA, USA) and donkey anti‐goatCy3 (Jackson) respectively, diluted 1:600 in PAD. For GLS2, the second incubation took 45 minutes at 37°C with goat anti‐rabbit Cy3, diluted 1:300 in PAD. After staining, sections were mounted in Fluoromount (SERVA Electrophoresis GmbH, Heidelberg, Germany).

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).