Tris buffered saline tween20 tbst

Western blots were performed on protein extracts collected in Laemlli Buffer (Sigma) supplemented with an additional 10% SDS to comply with local controls for the removal of samples from containment level 4. Samples were heated at 95 °C for 10–15 minutes prior to removal to containment level 2. Protein lysates were loaded on to Novex 4–12% gradient acrylamide gels (Life Technologies). The gels were run in a Novex X-2 vertical gel tank for 50 minutes at 200 V in MOPS buffer (Life Technologies). PVDF blots were performed using the IBlot semi-dry blotting system (Life Technologies) and blots blocked for minimum 2 h at room temperature with non-fat dried milk (NFDM; Sigma) in Tris-Buffered Saline–Tween20 (TBST; Sigma). Blots were rinsed in TBST and primary antibody staining performed overnight with the IRF-3 and GAPDH specific rabbit polyclonal antibodies (Millipore). Secondary antibody staining was performed as per antibody instructions using goat anti-rabbit antibody conjugated to HRP. Luminescence assay was performed with ECL Prime reagent (GE Healthcare) after a 5 minute incubation and imaged in a GeneSys Gel Imaging System (GeneSys).

Pretreated with cold phosphate-buffered saline (PBS), transfected cells were exposed to radioimmunoprecipitation assay (RIPA) lysis buffer added with 1% protease inhibitor (Sigma-Aldrich) for protein extraction. Protein concentration was assessed with the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples at 40 μL/lane were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). Then, the separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Following an incubation with 5% skim milk powder, the membrane was incubated overnight at 4°C using target primary antibodies, followed by 2 h of hybridization with horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG. Tris-Buffered Saline Tween-20 (TBST) (Sigma-Aldrich) was used to wash the membrane three times. Protein bands were detected with electrochemiluminescence (ECL; Beyotime, Shanghai, China), and quantitative analysis was conducted with Image Lab (Bio-Rad). The experiment was repeated 3 times. (Antibody information is shown in Table 2.)

Blood samples (12 mL divided into 2 tubes of 6 mL each) were taken by venipuncture and collected in a heparin tube after a resting period of 30 min at room temperature (25°C) and 1 min post-effort. The blood was placed on ice to be centrifuged at 3000 g for 15 min at 4°C) and the plasma was extracted from a tube and stored in an Eppendorf freezer tube (Invitrogen, USA) at –80°C for further analysis. The plasma BDNF analysis was performed using the ELISA kit E-max^® (Promega, USA) according to manufacturer's recommendations. Samples from the studied groups previously stored at –80°C were centrifuged for 5 min at 1500 g at 4°C and the supernatant was transferred to a 96-well plate (Corning Costar, USA), coated with anti-BDNF (1:1000), and then incubated for 2 h at room temperature. After this period, the plate was washed with Tris-buffered saline Tween-20 (TBS-T) (Sigma, USA) and incubated with the following antibodies: anti-human (1:500) (Promega, USA) for 2 h and conjugate anti-IgY HRP (1:200) for 1 hour. After these procedures, color reaction with tetramethyl benzidine was quantified in a plate reader at 450 nm (Quick Elisa, Thermo Fisher Scientific, USA). The values are reported in pg/mL.