Ab180714

Immunohistochemistry was carried out following the manufacturer’s recommendations. Tissue sections were embedded in paraffin and deparaffinized with xylene for 15 min, fixed with 100% ethanol for 10 min, and then rehydrated. The prepared slices were treated with methanol solution containing 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Sections were washed twice with PBS and then incubated with antibodies against Snail, E-cadherin, Slug, ZEB1, Twist, Vimentin and Survivin overnight (dilutions 1:200, 1:100, 1:100, 1:50, 1:75, 1:200, and 1:100) at 4°C. All sections were subjected to a heat-induced antigen retrieval process. Sections were again washed with PBS twice and then incubated with the corresponding secondary antibody under ambient conditions for 30 min. Then, after color development with 3-amino9-ethylcarbazole for 15 min, the slides were counterstained with hematoxylin. Finally, an optical microscope was used to observe the slides. Negative and positive controls were used to optimize staining. EMT inducers (Snail, Slug, ZEB1, and Twist) and EMT markers (E-cadherin, Vimentin, and Survivin) were also used. Rabbit anti-human polyclonal antibodies against Snail (ab180714), E-cadherin (ab15148), Slug (ab180714), ZEB1 (ab87280), Twist (ab49254), Vimentin (ab45939), and Survivin (ab469) were purchased from Abcam (Cambridge, United Kingdom).

Total protein was extracted from cells. Protein concentration was measured using a bicinchoninic acid kit (Thermo). Protein (30 μg) was subjected to polyacrylamide gel electrophoresis at 80 V for 35 min and 120 V for 45 min. After the electrophoresis was completed, proteins were transferred to a polyvinylidene fluoride membrane. Membrane was blocked by 5% skim milk at ambient temperature for 1 h. Membranes were incubated with rabbit anti-FOXK1 (1:1,000, ab18196; Abcam), rabbit anti-Snail (1:1,000, ab180714; Abcam), rabbit anti-MMP-2 (1:1,000, ab97779; Abcam), rabbit anti-MMP-9 (1:1,000, ab73734; Abcam), rabbit anti-Bcl-2 (1:2,000, ab182858; Abcam), rabbit anti-Bax (1:2,000, ab32503; Abcam), rabbit anti-E-cadherin (1:500,000, ab76319; Abcam), rabbit anti-N-cadherin (1:2,000, ab18203; Abcam), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2,500, ab9485; Abcam) at 4°C overnight. Membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:10,000, ab6721; Abcam) for 1 h at ambient temperature after PBST washing. Membranes were washed three times with PBST buffer for 10 min each. After development with an optical luminometer (GE, Boston, MA, USA), gray intensity of protein bands was measured by Image ProPlus 6.0 (Media Cybernetics, Rockville, MD, USA).

Sections were dewaxed with xylene and dehydrated with gradient alcohol. Sections were heated with 0.01 M citrate solution and allowed to cool down to room temperature after 20 min. After being maintained with 0.01 M PBS for 5 min, 3% hydrogen peroxide was added for 15 min to remove endogenous peroxidase. After PBS washing for 15 min, the sections were blocked with goat serum at 37°C for 20 min. The sections were incubated with primary rabbit anti-FOXK1 (1:1,000, ab18196; Abcam), rabbit anti-Snail (1:1,000, ab180714; Abcam), rabbit anti-MMP-2 (1:1,000, ab37150; Abcam), and rabbit anti-E-cadherin (1:500,000, ab76319; Abcam) overnight at 4°C. PBS served as NC instead of primary antibody. Sections were washed three times with PBS and incubated with goat anti-rabbit IgG (1:1,000, ab150117; Abcam) at 37°C for 30 min. Sections were rinsed with PBS for 15 min and incubated with streptavidin-biotin complex (Boster, Wuhan, P.R. China) at 37°C for 30 min. Color was developed by 3,3′-diaminobenzidine (DAB), followed by staining with hematoxylin for 1 min. Sections were washed with water for 1 min, followed by reaction in 1% hydrochloric acid and washing under running water. Sections were dehydrated and stained with saturated aluminum carbonate for 30 s, rinsed for 1 min, and treated with xylene for 15 min.