Peroxidase conjugated anti mouse or anti rabbit antibody

To obtain whole cell lysates, cells were extracted six days after transduction by use of Mammalian Protein Extraction Reagent (M-PER, Thermo Fisher Scientific) and subjected to at least three freeze-thaw cycles (− 80 °C/room temperature). Samples were run on a sodium-dodecyl sulfate/ 10% polyacrylamide gel and blotted onto a Westran S polyvinylidene difluoride membrane (GE Health Care). After blocking for 24 h in a 5% milk solution, proteins of interest were detected by incubation for 24 h with antibodies against MAP3K7, NF-κB p100/p52, NF-κB p105/p50, IκB (all Cell Signaling) used at a 1:1000 dilution in 5% milk/TBS-T (Tris-buffered saline with 0.5% Tween-20) or 5% BSA (bovine serum albumin)/TBS-T for phospho NF-κB p65 Ser536 (Cell Signaling). Incubation for 1 h with a peroxidase-conjugated anti-mouse or anti-rabbit antibody (both Sigma) was followed by signal detection with Western Lightning Plus-ECL detection reagent (PerkinElmer) using the Fusion FX detection system (Vilber Lourmat). ImageJ (1.48v) was used for quantification of results.

Consecutive 3-µm sections were cut from every block, and an immunoperoxidase technique was implemented following antigen retrieval by either microwave treatment (95 °C), pepsin (DAKO; Agilent Technologies, Inc., Santa Clara, CA, USA) treatment for 20 min and/or citrate buffer (pH 6.0) treatment for 45 min. Following endogenous peroxidase blocking with 3% H₂O₂-methanol for 15 min, the specimens were rinsed with phosphate-buffered saline (PBS) for 3 times. Anti-TrkA, anti-TrkB and anti-TrkC antibodies (Abcam, Cambridge, UK) were diluted to 0.5 µg/mL and were used as primary antibodies. Following 12 h incubation at room temperature, the specimens were rinsed with PBS three times and treated for an hour at room temperature with the secondary antibody [peroxidase-conjugated anti-mouse or anti-rabbit antibody (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)] that was diluted to 0.5%. The probes were subsequently rinsed with PBS 3 times and staining was performed using diaminobenzidine (DAB) solution (DAKO). Following drying, the samples were counterstained with Meyer hematoxylin (Sigma-Aldrich). The immunostaining of all samples was conducted under the same conditions as the antibody reaction and DAB exposure. The evaluation of Trk expression was performed by observing 20 microscope fields at ×100 magnification (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Equivalent amounts of eluted proteins were run on a 12% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, Burlington, MA, USA) using a semi-dry Trans-Blot Turbo Transfer System (BioRad, Irvine CA, USA). The membrane was incubated with a blocking buffer (TBS containing 5% BSA and 0.05% Tween-20) for 1 h at room temperature. Incubations with the diluted primary antibody in TBS 0.05% Tween-20 were performed at 4 °C overnight. After 2 h incubation with an anti-rabbit or anti-mouse peroxidase-conjugated antibody (1/5000, Sigma-Aldrich) at room temperature, the membrane was washed five times (5 min each) with PBST. The membrane was then treated with ImmunoCruz™ Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions.