Bronchi

Of the 199 samples of primary lung carcinoma obtained by surgery in the Saitama Medical Center, 100 samples which had been preserved and fixed at a high quality, and demonstrated no necrosis or degeneration and positive staining in bronchial gland cells, were prepared for IHC by paraffin section. These 100 samples consisted of 73 AC, 23 SCC, 1 LCC, 2 SmCC and 1 pleomorphic carcinoma case (Table I). Tissues from pathological lesions, together with adjacent normal tissues were fixed in 10% formaldehyde at room temperature for 24 h. Tissues were sliced into small pieces, embedded in paraffin and cut into 3.5 µm sections. Heat-induced epitope was performed using PT Link (cat. no. PT100/PT101; Agilent Technologies, Inc.) and EnVision^TM FLEX Target Retrieval Solution, High pH (pH 9.0; cat. no. S2367; Agilent Technologies, Inc.) for TFF1 and TFF3, and Low pH (pH 6.0; cat. no. #S2031; Agilent Technologies, Inc.) for TFF-2 at 95°C for 30 min. As the antibodies used for ELISA did not react in paraffin-embedded tissues, commercialized primary antibodies as follows were used: Anti-TFF1 rabbit polyclonal (1:350; cat. no. PA5-31863; Invitrogen; Thermo Fisher Scientific, Inc.), anti-TFF2, rabbit polyclonal (1:50; cat. no. ab131147; Abcam) and anti-TFF3 rabbit monoclonal (1:300; cat. no. ab108599; Abcam). Sections were incubated with the blocking solution included in the kit for 20 min at room temperature, with primary antibodies at 4°C overnight and with secondary antibodies for 20 min at room temperature and visualization was performed with a Catalyzed Signal Amplification System II kit which included secondary antibodies (cat. no. K1497 and K1501; Agilent Technologies, Inc.). A total of 5 high magnification fields of view were randomly selected from each section and 200 cells were counted per field. Images were digitally captured using a BX51 bright-field microscope (Olympus Corporation) equipped with a DP-72 digital camera in conjunction with the cellSens imaging software (version 1.18, Olympus Corporation). Adobe Photoshop was used for image processing (CS4 Extended; Adobe Systems Europe, Ltd.)
IHC score was determined semi-quantitatively by multiplication of the ‘positive fraction’ with the ‘intensity-score’ according to the following system. Firstly, the positive fraction was sub-divided as follows: i) 0, no staining; ii) 1, <10% staining; iii) 2, 10–50% staining; and iv) 3, >50% staining of cells with intensity score >0. Secondly, intensity-score was determined as follows: i) 0 if there was no staining or staining was weaker than that in non-neoplastic cells; ii) 1 if the staining was the same as in non-neoplastic cells; and iii) 2 if there was more intense staining than in the non-neoplastic cells. An IHC score >0 was defined as ‘positive’ (44 (link)). Staining was evaluated by two observers and discordance was resolved by discussion.