Anti bdnf

Proteins were extracted from brain samples using lysis buffer (Pierce Ripa buffer, Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL, USA).
From each sample, 30 µg of total proteins were denatured in 5 × Laemmli sample buffer and loaded into Any Kd pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for Western blot analysis. Anti-BDNF 1:250 (MYBioSource, San Diego, CA, USA), anti-TrkB 1:1000, anti p75 NTR 1:500 (Thermo Scientific, Rockford, IL, USA) and β-actin 1:2000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. After overnight incubation, the membranes were further incubated with a horseradish peroxidase-conjugated rabbit secondary antibody (Bio-Rad, Milan, Italy). The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA) and immunoreactive bands were quantified using Image Lab Software (BioRad Laboratories Inc., Hercules, CA, USA) and normalized against β-actin expression.

Protein extracts were obtained using a standard procedure from colon and brain samples of Ctrl-Std, KD, IBS-Std, and IBS-KD rats. For Western blot analysis, the aliquots of 50 µg of total protein extracts from each sample were loaded into 10% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy). Anti-5-HT_3B, anti-5-HT₄, anti-SERT (Abcam, Cambridge, UK), anti-BDNF (MyBioSource, San Diego, CA, USA), anti-TrkB (Thermo Scientific, Rockford, IL, USA), anti-β-actin, and anti-GAPDH (Cell Signaling, Danvers, MA, USA) were used as primary antibodies. The proteins were detected by chemiluminescence (ECL, Bio-Rad, Milan, Italy), and the densitometric analysis of each protein-related signal was obtained using the Molecular Imager Chemidoc^TM (Bio-Rad, Milan, Italy) and normalized against β-actin or GAPDH expression.