Chemidoc image detector

Brain tissue was homogenized in chilled cell lysis buffer (0.5% NP-40, 0.5% Triton X-100, 0.1% sodium deoxycholate, 50 mM Tris-HCl, 150 mM sodium chloride, and 1 mM EDTA) containing protein inhibitors. The dissolved proteins were left on ice for 40 min and then collected after centrifugation at 12,000× g for 20 min at 4 °C. Protein concentration was determined using a bicinchoninic acid assay (BCA) (GenDEPOT, Barker, TX, USA). A quantity of 5–20 μg total protein was loaded onto a 4–20% gel for SDS-PAGE, transferred to a PVDF membrane, blocked with skimmed milk for 1 h, and then incubated overnight with primary antibodies against the following proteins at 4 °C: BDNF, Bcl-2, Bax, Caspase-3, PARP, and β-actin (Abcam, Cambridge, MA, USA). The membranes were then incubated with secondary antibodies for 1 h and developed using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) and a ChemiDoc image detector (Bio-Rad, Hercules, CA, USA).

The LPS (1 μg/mL) or anti-DNP IgE (200 ng/mL) and SBP extract-treated cells were lysed in a lysis buffer (50 mM Tris-hydrochloride (HCl), 150 mM sodium chloride, 0.5% Triton X-100, 0.5% NP-40, 0.1% sodium deoxycholate, and 1 mM EDTA) for 40 min on the ice. Then, the supernatant was collected through centrifugation (12,000× g, 4 °C, 20 min). Proteins were separated via 4–20% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with skim milk for 1 h and then incubated overnight at 4 °C with appropriate primary antibodies (iNOS, COX-2, STAT1, p-STAT1, T-bet, interferon regulatory factor 1 (IRF1), STAT6, p-STAT6, GATA binding protein 3 (GATA3), c-maf, and β-actin). After incubation, the cells were washed with 1× TBST and incubated with the secondary antibody for 1 h. Enhanced chemiluminescence was used to develop protein bands, and the signal was detected using a Chemi-doc image detector (Bio-Rad, Hercules, CA, USA).