Alexa 350

The processing of the bladder samples was carried out at the A*STAR/IMCB histology facility (A*STAR, Singapore). Briefly, the bladder tissues were embedded in 2% agar for paraffin processing. For IFA, 4- to 5μm serial sections were cut longitudinally, deparaffinized in xylene (twice for 5 min at room temperature), rehydrated in 100% ethanol (twice for 2 min at room temperature), 95% ethanol (for 1minute), and 80% ethanol (for 1 minute). Antigen retrieval was performed by boiling slides for 30 min in 1mM EDTA buffer, pH 8. Slides were blocked in 1% FBS 0.4% TritonX-100 for 1 h at room temperature, and subsequently incubated overnight at 4°C with the following primary antibodies- goat anti GFP antibody (Abcam 1:500 dilution in blocking buffer) and rabbit anti Rab35 antibody (Novus biological, 1in 50 dilution in blocking buffer). For LAMP1 triple staining, the sections were additionally stained with rat anti LAMP1 antibody (abcam 1:200 blocking buffer). Sections were washed thrice with blocking buffer and were subsequently incubated with Alexa Fluor 488 (anti-rabbit), Alexa 594 (anti-goat) and Alexa 350 (anti-rat) conjugated secondary antibodies (1:500; Molecular Probes). The sections were then stained with DAPI and were analyzed by confocal microscopy.

To visualize mRNA using the red channel, after fixation HeLa cells were incubated with oligo-dT-[Cy3], diluted in SSC 2×, 1 mg/ml yeast tRNA, 0.005% BSA, 10% dextran sulfate, 25% formamide, for 2 h at 37°C. Wash steps were performed using 4× and then 2× SSC buffer (0.88% sodium citrate, 1.75% NaCl, pH 7.0). To visualize mRNA in blue color for SGs experiments, the oligo-dT with digoxigenin was used after cells fixation with the same incubation procedure as oligo-dT-Cy3. Then the primary anti-digoxigenin antibodies (mouse, ab420, Abcam) and secondary antibodies (goat anti-mouse, Alexa 350, Invitrogen) were applied to cells according to supplier's protocol.

Cultures were fixed in 4% paraformaldehyde (PFA), 4% sucrose for 15 minutes at room temperature. Cells were then washed twice with PBS for 5 min and permeabilized for 45 min with 0.2% Triton X-100 and 1% BSA in PBS. Primary antibodies were then added and the samples incubated at 4 °C overnight in PBS. The samples were rinsed twice for 5 minutes with PBS and further incubated with the corresponding secondary antibodies for 2 hours at room temperature. The chips were then rinsed once with PBS and once with PBS + 0.1% sodium-azide. The following primary antibodies were used: VGLUT1 (gift from Dr. Salah El Mestikawy, IBPS CNRS UMR8246, Paris, France, 1/1000), MAP2 (M4403, Sigma, 1/500). Species-specific secondary antibodies coupled to Alexa 350, 488, 555 or 633 were used (1/500, Invitrogen) to visualize bound primary antibodies.

To evaluate the integrity of the primary hippocampal neurons inside our microfluidic chips, some chips were fixed in PBS containing 4% paraformaldehyde (PFA, Electron Microscopy Science 15714S) and 4% sucrose (Sigma S9378) for 8 min at room temperature and rinsed twice in PBS for 5 min. Then, they were permeabilized for 30 min at room temperature with 0.2% Triton (Sigma X100) and 1% bovine serum albumin (BSA, Thermofisher 15561020) in PBS. After removing the permeabilizing solution, primary antibodies were added in PBS with 1% BSA and the samples incubated at 4 °C overnight. The samples were rinsed for 5 min with 1% BSA in PBS and further incubated with the corresponding secondary antibodies for 2 h in PBS with 1% BSA at room temperature. Finally, the chips were rinsed first with 1% BSA in PBS and last only with PBS. The following primary antibodies and dye were used: DAPI (4’,6-diamidino-2-phénylindole, nuclear DNA staining) (1050, Euromedex, 1/2000), MAP2 (Microtubule-associated protein 2) (M4403, Sigma, 1/1000), β3-tubulin (MA1-118, Thermofisher, 1/1000) and GFAP (Glial Fibrillary Acidic Protein) (Z0334, Dako, 1/1000). Species-specific secondary antibodies coupled to Alexa 350, 488, 555 or 633 were used (1/1000, Invitrogen) to visualize bound primary antibodies.

All chemicals were obtained from Sigma (St Louis, MO) or Fisher (Waltham, MA) unless otherwise stated. [9,10-³H]oleic acid (sp act: 50 Ci/mmol) was purchased from Moravek (Brea, CA), [³H]uracil from PerkinElmer (Shelton, CT), 5-Butyl-4,4-Difluoro-4-Bora-3a,4a-Diaza-s-Indacene-3-Nonanoic Acid (C4-BODIPY-C9) from ThermoFisher Scientific (Waltham, MA) and 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene (BODIPY 493/503) from Life Technologies (Carlsbad, CA). Non-polar lipid mixture for thin-layer chromatography was purchased from Matreya LLC (State College, PA). Atglistatin was purchased from Cayman Chemical (Ann Arbor, MI). The primary antibodies include: rabbit polyclonal anti-GRA7 [34 (link)], the rabbit polyclonal anti-aldolase and mouse monoclonal anti-Hsp70 (from Fidel Zavala, Johns Hopkins university, Baltimore), and the mouse monoclonal anti-SAG1 (from Jean-Francois Dubremetz, University of Montpellier, France). Secondary antibodies used for immunofluorescence were conjugated to Alexa⁴⁸⁸, Alexa⁵⁹⁴ or Alexa³⁵⁰ (Invitrogen, Carlsbad, CA). To prepare oleic acid (OA)-albumin complexes, sodium oleate was dissolved in H₂O at a concentration of 100 mM, then thoroughly mixed by vortexing for 3 min with 5% Fatty Acid Free BSA to reach a final concentration of 10 mM OA-BSA complexes.