Dab developing solution

EML4-ALK fusions were independently confirmed by FISH and IHC analysis of 4μM serial sections of FPPE fixed tissue. FISH was performed using the Vysis ALK Break Apart FISH Probe Kit (Abbott, Singapore) according to the supplier's protocol. For IHC, immunoreactive ALK antigen was retrieved by incubation in 0.01 mol/L citrate buffer solution (pH 6.0) at 100°C for 10 min. Endogenous peroxidase activity was inactivated by further incubation in 3% hydrogen peroxide for 10 min. ALK fusion protein was finally detected by incubation with anti-ALK (D5F3) rabbit MAb (Cell Signalling Technology, US) followed by a secondary mouse anti rabbit HRP-linked IgG antibody (Dako REAL™ EnVision™/HRP, Rabbit/Mouse detection system). ALK fusion protein staining was finally revealed using the DAB developing solution (Dako Cytomation, Glostrop, Denmark).

The degree of extracellular matrix deposition was assessed via immunohistochemical analysis using the EnVisionTM Detection Systems Peroxidase/DAB, Rabbit/Mouse (Dako, Cambridgeshire, UK) kit. Briefly, sections were incubated with ‘Dual Endogenous Enzyme Block’ from the EnVisionTM kit for 10 min before blocking with 20% normal goat serum (Dako, Cambridgeshire, UK) in PBS for 30 min. After being washed in PBS for 5 min, the primary antibodies (collagen type I: Col1a, 1:100 dilution and osteocalcin: OCN, 1:800 dilution) (Abcam, Cambridge, UK) were added to samples at the desired concentration in 1% BSA (Sigma-Aldrich, Gillingham, UK) in PBS and left to incubate overnight at 4 °C. Then, the sections were washed in PBS for 10 min, followed by incubation in the secondary HRP goat anti-rabbit IgG antibody for 30 min. After that, the Dako DAB developing solution was applied to slides for 10 min and counter stained with Harris Haematoxylin before being visualised under an Olympus BX50 microscope.

The mouse intestinal tight junction (TJ) proteins (ZO-1 and Occludin) were stained. To be specific, the tissue sections were baked in the oven at 60° C, deparaffinized with xylene, and dehydrated with gradient alcohol, followed by antigen retrieval in the microwave at 98° C for 20 min. Later, the sections were incubated with 3% hydrogen peroxide at room temperature for 10 min to eliminate the endogenous peroxidase. After blocking with 2% bovine serum albumin (BSA) at room temperature for 30 min, the sections were incubated with monoclonal antibodies against ZO-1 and Occludin (Abcam, MA, USA; diluted with TBST at 1:500) at 4° C overnight. Thereafter, the sections were further incubated with IgG secondary antibody at 37° C for 15 min, and then with peroxidase-labeled streptomycin (Maixin Biotechnology Co., Ltd; Fuzhou, China) for another 15 min. Afterwards, the sections were washed with PBS thrice, with 5 min each time. Later, each section was dropwise added with freshly prepared DAB developing solution (DAKO, Denmark), counter-stained with hematoxylin and mounted. The negative control primary antibody was replaced by TBST. The Olympus-DP72 image collection system and the Olympus-BX51 upright microscope of the CRi Nauance multispectral imaging system were adopted for photographing and analysis.