The membrane was probed with an anti-SOX9 rabbit antibody (1:2,000 dilution; Millipore) and incubated with goat anti-rabbit immunoglobulin G (1:50,000 dilution; Pierce). Expression of SOX9 was determined with SuperSignal click here West Pico Chemiluminescent Substrate (Thermo,

USA) according to the manufacturer’s suggested protocol. The membranes were stripped and reprobed with an anti-actin mouse monoclonal antibody (1:2,000 dilution; Millipore) as a loading control. Immunohistochemistry (IHC) Immunohistochemical analysis was performed to study altered protein expression in 142 human lung cancer tissues. The procedures were carried out in a similar manner to previously described methods [13]. Paraffin-embedded specimens were cut into 4 μm sections and baked learn more at 65°C for 30 minutes. The sections were deparaffinized with xylenes and rehydrated. Sections were submerged into ethylenediaminetetraacetic acid antigenic retrieval buffer and microwaved for antigenic retrieval. The sections were treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation in 1% bovine serum albumin to block non-specific binding. Rabbit anti-SOX9 (1:50 dilution; Millipore) was incubated with

the sections at 4°C overnight. see more Primary antibody was replaced by normal goat serum in the negative controls. After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody (Zymed, San Francisco, USA) followed by a further incubation with streptavidin-horseradish

peroxidase complex (Zymed). The tissue sections were immersed in 3-amino-9-ethyl carbazole and counterstained using 10% Mayer’s hematoxylin, dehydrated, and mounted in Crystal Mount (Sigma). The degree of immunostaining of formalin-fixed, paraffin-embedded sections was viewed and scored separately by two independent investigators, who were blinded to the histopathological features and patient details of the samples. Scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The scores given by the two independent investigators were averaged for further comparative evaluation of SOX9 expression. The proportion of positively stained tumor cells was staged triclocarban as follows: 0 (no positive tumor cells), 1 (<10% positive tumor cells), 2 (10-50% positive tumor cells), and 3 (>50% positive tumor cells). The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). The staining index was calculated as follows: staining index = staining intensity × proportion of positively stained tumor cells. Using this method of assessment, the expression of SOX9 in lung cancers was evaluated using the staining index (scored as 0, 1, 2, 3, 4, 6, or 9).