Hrp conjugated goat anti mouse igg secondary antibody

The 5 μm thick paraffin-embedded tissue sections were cut onto poly-L-lysine-coated microscope slides (ThermoFisher Scientific, Waltham, MA, USA), deparaffinized, and rehydrated. Sections were incubated for 20 min at 95 °C with 0.01 M citrate buffer (pH 6.0) for antigen retrieval. Sections were stained using an autostainer (DAKO, Glostrup, Denmark) with 2 μg/ml of anti-EpCAM antibody for 30 min at RT. Sections were then incubated for 1 h at RT with HRP-conjugated goat anti-mouse IgG secondary antibody (DAKO, Glostrup, Denmark) and developed for 5 min with 3,3-diaminobenzidine (ThermoFisher Scientific, Waltham, MA, USA), followed by counterstaining using hematoxylin. Colon cancer tissue sections known to express EpCAM were used as a positive control in each batch of IHC staining.

Cell lysates were collected in ice-cold lysis buffer consisting of TNE (Tris-NaCl EDTA) buffer, pH 7.5, and 1% Nonidet P-40 (catalog number 37129000; Roche) in the presence of the protease inhibitor cocktail (cOmplete mini EDTA free; catalog number 11836170001; Roche). The procedure for SDS-PAGE and Western blotting is extensively described in Deruelle et al. (47 (link)). Blots were blocked in 5% (wt/vol) nonfat dry milk diluted in 0.1% phosphate-buffered saline (PBS)-Tween 20 (PBS-T) for 1 h at room temperature. The primary antibodies, the mouse monoclonal anti-IκBα antibody (clone L35A5; 1:1,000 dilution; catalog number 4814; Cell Signaling Technology), and the mouse monoclonal horseradish peroxidase (HRP)-conjugated anti-α-tubulin antibody (clone DM1A; 1:2,000 dilution; catalog number ab40742; Abcam) were incubated overnight at 4°C diluted in blocking buffer. After three 10-min washing steps with PBS-T, IκBα blots were incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:2,000 dilution; catalog number P0447; Dako) for 1 h at room temperature. Protein bands were visualized via chemiluminescence, using either ECL Plus substrate (GE Healthcare) or SuperSignal West Femto maximum-sensitivity substrate (Thermo Scientific), with a ChemiDoc MP imaging device (Bio-Rad).