Amersham ecl reagant

In dissected DRG or nerve, MCT1 mRNA was isolated by RNeasy Mini Kit (Qiagen), reverse transcribed to cDNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and quantified by real-time RT PCR using Taqman probes (Applied Biosystems) for MCT1 relative to 18sRNA (DRG), actin, or peptidylprolyl isomerase A (PPIA; nerves) on a StepOne Plus RT-PCR System (Applied Biosystems). These housekeeping genes were chosen based on preliminary and/or published data (Girolami, Bouhy et al. 2010 (link)) that demonstrated gene expression to be stable following nerve crush. For Western blots, sciatic nerves, either proximal or distal to the crush, were homogenized in T-PER (Thermo Scientific) and run on Mini-Protean TGX Gels (10%; Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). For all Western blots, 5 μg of sciatic nerves and 20 μg of brain were run on the gel. Membranes were incubated overnight with Rabbit polyclonal β-actin antibodies (AbCam; 1:3000) and visualized with Amersham ECL Reagant (GE Healthcare) on ImageQuant LAS 4000 (GE Healthcare). After visualizing β-actin, blots were stripped with Restore Western Blot Stripping Buffer (Thermo Scientific), reprobed overnight with chicken antibodies specific for mouse MCT1 (Rothstein laboratory; 1:300), and again visualized by ECL reagent, as described above.

Peripheral nerves dissected after transcardial perfusion of deeply anesthetized mice with 0.1 M PBS were homogenized in T-PER (Thermo Scientific) and run on Mini-Protean TGX Gels (10%; Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). For all Western blots, 15–30 μg of proteins were separated on the gel. Membranes were incubated overnight with MCT1 (generated for laboratory (Morrison et al., 2015 (link)); 1:200), P0 (Aves Labs. Inc.; catalog # PZO; myelin protein-zero chicken polyclonal anti-peptide antibody; 1:4,000), MBP (Millipore Sigma; catalog # AB980; anti-myelin basic protein antibody; 1:250), or MAG (Millipore Sigma; catalog # PA5–30087; polyclonal antibody; 1:500) antibodies and visualized with Amersham ECL Reagant (GE Healthcare) on ImageQuant LAS 4000 (GE Healthcare). After visualizing for above primary antibodies, blots were stripped with Restore Western Blot Stripping Buffer (Thermo Scientific), reprobed overnight with β-actin (Millipore Sigma; catalog # A5316; monoclonal anti-β-actin antibody; 1:5,000), and again visualized by ECL reagent, as described above.