Horseradish peroxidase hrp conjugated secondary antibody

Total proteins were extracted using Buffer C lysis buffer. The protein concentrations were measured by bicinchoninic acid (BCA) assay. Forty micrograms of proteins were concentrated at 80 mV and separated at 100 mV using 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA) for 2 h at 300 mA following the electrophoresis. Membranes were blocked with 10% milk at room temperature for 1 h and incubated with the following primary antibodies: c-fos (sc-413, mouse monoclonal, Santa Cruz CA), BDNF (ab226843, rabbit polyclonal antibody; abcome echnology), CREB and phospho-CREB (sc-377154, sc-81486, mouse monoclonal, Santa Cruz, CA) at 4°C overnight. Membranes were washed 3 times (10 min × 3) with Tris-buffered saline-Tween (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Applygen, Beijing, China) for 1 h at room temperature followed by washing 3 times (10 min × 3) with Tris-buffered saline-Tween (TBST). Proteins were developed using an enhanced chemiluminescence (ECL) reagent kit (Applygen, Beijing, China) and radiographic films (Carestream, Xiamen, China). α-tubulin (ABT170, rabbit polyclonal, 1:10,000, Millipore, Temecula, USA) was used as the internal reference.

As described previously (Wang et al., 2017 (link)), cells were harvested by Western blot to analyze the abundance of the protein. Membranes were incubated with primary antibodies [occludin, claudin-1, ZO-1, P38, p-P38 and NF-κB (P65, P-p65) (Santa Cruz Biotechnology, USA)] overnight at 4°C and then washed. TBST 3 times in 15 min. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Applygen Technology, Inc., Beijing, China) for 1 h at room temperature. Signals were detected using the ImageQuant LAS 4000 mini-system (GE Healthcare Bio-sciences AB, Inc., Sweden) using Western Blot Brightness Reagent (Applygen, Beijing, China) and passed through Image Quant TL Software (GE Healthcare Life Science) for the gel imaging system.
The expression levels of intercellular tight junction proteins (TJPs) occludin, and claudin-1 were evaluated by immunofluorescence microscopy as previously described (Qin et al., 2009 (link); Donato et al., 2010 (link)). Briefly, IPEC-J2 cells were incubated with a rabbit anti-occludin Ab and a rabbit anti-claudin-1Ab (Abcam, USA) and then with FITC-conjugated goat anti-rabbit secondary Ab. After washing with PBS, Cells were removed from the plastic support, mounted on glass slides with Vectashield containing DAPI, and examined on a Leica TCS SP5 confocal laser microscope (Keyence, Osaka, Japan).