Gtx125918

Formalin-fixed and paraffin-embedded tissues from lungs of SCID mice were evaluated by hematoxylin and eosin (H & E) staining and immunohistochemistry using polyclonal antibodies against LCN2 (Epitomics; #3639–1), Snail (GeneTex; GTX125918), N-cadherin (GeneTex; GTX112734), and fibronectin (GeneTex; GTX112794) using the avidin-biotin complex method. Positive staining of cancer cells for LCN2 was identified by a dark brown color indicative of LCN2 immunoreactivity. The staining intensity was graded as absent (0), weak (1+), medium (2+), or strong (3+). The histoscore (Q) was calculated by multiplying the percentage (P) of positive cells by the intensity (I), according to the formula: Q = P × I. The mean Q of each type of cervical cancer was chosen as previously described [20 (link), 21 (link)].

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors for 20 min. Proteins (20 μg) extracted from cells were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with blocking buffer (5% non-fat milk in PBS containing 0.1% Tween 20) and then incubated with primary antibodies dissolved in blocking buffer at 4 ℃ overnight. Membranes were washed with TBS-T buffer (PBS with 0.1% Tween 20) for 5 min three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. The membrane was developed using an ECL Kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) with X-ray film.
The primary antibodies used in this western blotting were as follows: E-cadherin (H-108, 1:1000, sc-7870; Santa Cruz Biotechnology, Inc.), N-cadherin (H-63, 1:1000, sc-7939; Santa Cruz Biotechnology, Inc.), fibronectin (EP5, 1:1000, sc-8422; Santa Cruz Biotechnology, Inc.), vimentin (V9, 1:1000, sc-6260; Santa Cruz Biotechnology, Inc.), slug (1:1000, GTX31749; GeneTex), snail (1:10000, GTX125918; GeneTex), and GAPDH (D4C6R, 1:1000, 97,166; Cell Signaling Technology).