Dab color development kit

Sixty male New Zealand large white male rabbits (2.5–2.8 kg) were provided by Experimental Animal Center of Dalian Medical University. Metoprolol was purchased from AstraZeneca plc (batch number: H10950118). Mouse anti-rabbit Cz43 (dilution, 1:500; cat. no. ab79010) and GAPDH monoclonal primary antibody (dilution, 1:500; cat. no. ab9485), goat anti-mouse horseradish labeled polyclonal secondary antibody (dilution, 1:2,000; cat. no. xab6789) were purchased from Abcam (Cambridge, MA, USA). DAB color development kit and hematoxylin were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China. TRIzol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Reverse transcription kit and PCR amplification kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). This study was approved by the Ethics Committee of Tianjin Third Central Hospital, Tianjin, China.

Tissue sections with a 4-μm thickness were made from paraffin-embedded lung specimens. The sections were then processed through dewaxing and antigen retrieval by citrate buffer (pH 6.0) for 15 min with the temperature in the range of 92°C–98°C. The sections were incubated with the primary antibody anti-ADAM9 IgG of a rabbit (Abcam, Cambridge, MA). Visualization was then performed with an EnVision detection system and a DAB color development kit (ZhongShan Golden Bridge Biotechnology, Beijing, China). The mean staining density of ADAM9 in the airway epithelium was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).