Ab28699

Protein samples from the cell lines and tissues were prepared with a lysis buffer, M-PER (PIERCE Biotechnology, Rockford, IL, USA), containing proteinase inhibitors. Total protein aliquots were mixed with the Laemmli sample buffer, denatured at 95°C for 5 min and separated on a 10% Tris-HCl gel (Bio-Rad). The proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% (w/v) nonfat milk/TBST for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies directed against HOXA11 (1:5000, ab28699, Abcam, USA) in 5% BSA/TBST (w/v). The membranes were washed three times with TBST and incubated with an HRP-linked secondary antibody for 1 h at room temperature. The protein bands were visualized using electro-chemiluminescence (ImageQuant LAS4000, USA). The anti-Tubulin antibodies served as the loading control.

Paraffin-embedded TMA blocks, including 160 lung adenocarcinoma samples (40 lung AIS, 40 lung AD and 80 normal counterparts), were sectioned at a 4 μm thickness, deparaffinized in xylene, and rehydrated in graded ethanol solutions, and the endogenous peroxidase activity was blocked by incubation with 3% H₂O₂ for 30 min at room temperature. Then, the sections were immersed in a citrate-NaOH buffer (10 mM sodium citrate, pH 7.0) for 40 min at 92°C to restore antigenicity. The rehydrated sections were incubated overnight at 4°C with the rabbit anti-human HOXA11 polyclonal antibody (1:250, ab28699, Abcam, USA.). The sections incubated with the first antibody were washed with Tris-buffered saline (TBS) and were then incubated with the MaxVision ^TM HRP-Polymer anti-Rabbit IHC Kit (Maixin, Fuzhou, China) for 15 min at room temperature. The sections were visualized using the DAB Detection Kit (Maixin, Fuzhou, China), and the reaction was followed by counterstaining with hematoxylin. The negative control experiments were performed by omitting the primary antibody.