Immunohistochemistry was performed on 4 µm thick sections by standard protocols with primary antibodies including TTF-1 (Novocastra, Great Britain), CK7 (Dako, Denmark), napsin A (Novocastra, Great Britain), CK5/6 (Dako, Denmark) and p63 (Novocastra, Great Britain).
The NEDD9 monoclonal antibody (Abcam, San Francisco, CA, USA) was used in 1:400 dilution with EnVision kit for visualization. Normal bronchial epithelium present in tumor surroundings was used as negative control. The ‘hot spot’ was determined by inspection on whole specimen at x40 magnification by two investigators blinded for the clinicopathologic data. The immunohistochemical reaction was examined at x400 magnification.
The percentage of NEDD9 positive tumor cells was assessed semiquantitatively, as follows: 0-25% of positive cells scored 0 or negative; 26%-50% as 1 (weakly positive); 51%-75% as 2 (mildly positive); and more than 75% of positive tumor cells as 3 (strongly positive). Positivity was evaluated separately for nuclei and cytoplasm. The intensity of staining was also scored in the range of 0-3 (negative, weakly stained, mildly or strongly stained) (Fig. 1 ).
The NEDD9 monoclonal antibody (Abcam, San Francisco, CA, USA) was used in 1:400 dilution with EnVision kit for visualization. Normal bronchial epithelium present in tumor surroundings was used as negative control. The ‘hot spot’ was determined by inspection on whole specimen at x40 magnification by two investigators blinded for the clinicopathologic data. The immunohistochemical reaction was examined at x400 magnification.
The percentage of NEDD9 positive tumor cells was assessed semiquantitatively, as follows: 0-25% of positive cells scored 0 or negative; 26%-50% as 1 (weakly positive); 51%-75% as 2 (mildly positive); and more than 75% of positive tumor cells as 3 (strongly positive). Positivity was evaluated separately for nuclei and cytoplasm. The intensity of staining was also scored in the range of 0-3 (negative, weakly stained, mildly or strongly stained) (