1d imaging analysis software

Near infrared fluorescent tracer injection was performed as described³⁰ (link)^,32 (link) with modification. Briefly, 100 μL of Rhodamine 800 (R800, 496 Da) at 1 mg/mL in PBS was injected intravenously into the tail vein in adult mice (6 months, WT or GPR110 KO). After 2 h the anesthetized animals were perfused for 5 min with Hanks’ balanced salt solution (HBSS). After perfusion, brains were removed and put on a 2 mm thick slide on ice. The accumulation of R800 dye was examined ex vivo using an Azure Sapphire Biomolecular Imager. Images were acquired was at several z-axis levels (ex784nm/λem823nm) and quantified with Kodak 1D imaging analysis software.

Samples were electrophoresed in 4-12% Bis-Tris gels at 200 V using MOPS SDS running buffer. Proteins were transferred to a PVDF membrane (Bio-Rad) at 25 V for 30 min using a Bio-Rad Trans-Blot Turbo transfer system. The membrane was blocked with 5% milk in TBS containing 0.1% Tween 20 (TBS-T) at room temperature for 1 h. Blots were washed three times with TBS-T, incubated with primary antibody (1/1000 dilution ratio, in TBS-T containing 5% BSA and 0.1% sodium azide) at 4°C overnight, washed three times with TBS-T, and incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing three times with TBS-T, blots were incubated with enhanced chemiluminescent (ECL) substrates for 5 min before imaged with a Kodak Gel Logic 440 Imaging system or Azura Sapphire Biomolecular Imager. Band intensity was quantitated using Kodak 1D imaging analysis software.