Immobilon fl polyvinylidene difluoride membrane

Brain tissues from mouse olfactory bulb and neocortex, naked mole rat neocortex and entire forebrain were homogenized in TNE buffer containing 0.5% Triton X-100 (Sigma), 1% octyl-β-D-glucopyranoside (Calbiochem), 5 mM NaF, 100 μM Na₃VO₄ and a cocktail of protease inhibitors (Complete^TM, Roche) by ultrasonication and lysed for 45 minutes at 4 ^oC. Cell debris and nuclei were removed by centrifugation (800 g, 10 min at 4 ^oC). Protein concentrations were determined by Bradford’s colorimetric method. Samples were diluted to a final protein concentration of 2 μg/μl, denatured in 5x Laemmli buffer, and analyzed by SDS-PAGE on 8% or 10% resolving gels. After transferring onto Immobilon-FL polyvinylidene difluoride membranes (Millipore), protein samples on membranes were blocked in Odyssey blocking buffer (Li-Cor Biosciences; 1 h) and exposed to primary antibodies (Supplementary Fig. 1b) overnight at 4 ^oC. Appropriate combinations of IRDye-800CW and IRDye-680-conjugated secondary antibodies were used for signal detection (Li-Cor Biosciences; from goat or rabbit hosts; 1:10,000, 2 h). Image acquisition and analysis were performed on a Li-Cor Odyssey IR imager. β-Actin (1:10,000; Sigma) served as loading control.

Hypothalamic lysate samples containing 40 µg of protein were separated on 4%–20% gradient polyacrylamide TGX gels (BIO-RAD, Hercules, CA, USA; 5671094) via electrophoresis and transferred to 0.45 µm pore size Immobilon-FL polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA; IPFL00010). Membranes were incubated in PBS-based blocking buffer (LI-COR Biosciences, Lincoln, NE, USA; P/N 927) for 1 h and then probed with primary antibodies overnight at 4 °C with shaking, followed by washing with PBS containing 0.01% Tween 20 (PBS-T). Blots were incubated with infrared fluorophore-bound secondary antibodies in the dark, washed again with PBS-T, and analyzed using the Odyssey CLx Imaging System (LI-COR Biosciences). After the phosphorylated signal transducer and activator of transcription 3 (pSTAT3) was measured, membranes were stripped (Re-Blot Plus Strong Solution; Millipore, Burlington MA, USA; 2504) and then probed for STAT3 to generate a pSTAT3/STAT3 ratio.

Protein from each sucrose gradient fraction (10 in total) obtained after exosome isolation were separated by SDS polyacrylamide gel electrophoresis[7] (link), [30] (link) using a XCell SureLock electrophoresis system (Life Technologies), transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and probed with primary mouse monoclonal anti-CD63 (1∶2,000 ab8219, Abcam, Sapphire Bioscience Pty Ltd, NSW, Australia), anti-CD81 (1∶1,500 MAB6435, Abnova, Tapei City, Taiwan) and anti-CD9 (1∶1,500 ab2215, Abcam, Sapphire Bioscience) as previously described exosome enriched markers. [7] (link), [18] (link), [29] , [30] (link) Membranes were washed in Tris buffer saline (pH 7.6) and incubated (1 h) in TBST/0.2% BSA containing horseradish peroxidase–conjugated goat anti-mouse antibody. Proteins were detected by enhanced chemiluminescence with using a SRX-101A Tabletop Processor (Konica Minolta, Ramsey, NJ, USA). The exosomal fractions (from 1.126 to 1.187 g/ml) were pooled (defined as exosomes) and placental alkaline phosphatase (PLAP) protein abundance was determined by Western blot using primary polyclonal antibody anti-PLAP (1∶1,000 ab96588, Abcam, Sapphire Bioscience) using a GS-800 Calibrated Densitometer (Bio-Rad Laboratories).