Immpress reagent

For the cross-sectional observations, tissues were fixed in 4% paraformaldehyde, processed, and embedded in paraffin. HE staining and Masson’s trichrome staining were performed using conventional methods. The following primary antibodies purchased from Abcam, Cambridge, UK were used. A monoclonal mouse anti-human serum albumin antibody (ab7793), a polyclonal rabbit anti-human CD31 antibody (ab28364), a monoclonal mouse anti-human CK18 antibody (ab82254), a monoclonal rabbit anti-human CK19 antibody(ab76539), a monoclonal rabbit anti-alpha Fetoprotein antibody (ab169552) and a monoclonal rabbit anti-human CYP3A4 antibody (ab124921). For immunohistochemistry, HRP detection was performed based on the micropolymer chemistry of the ImmPRESS™ Reagent (Vector Laboratories, Burlingame, CA, USA). FITC-conjugated goat anti-mouse IgG (H + L) (Invitrogen) and Alexa Fluor 555-conjugated goat anti-rabbit IgG (H + L) (Life Technologies;) or Opal 4-Color Manual IHC kit (Perkin Elmer, Waltham, MA, USA) were used for the immunofluorescence staining. Images were captured using a fluorescence microscope (BZ-X700, Keyence) and a confocal laser-scanning microscope (FV10i, Olympus).

The dissected lungs were filled with phosphate-buffered paraformaldehyde via intratracheal instillation and further fixed in phosphate-buffered paraformaldehyde. The lung tissues were embedded in paraffin, sectioned and subjected to staining with hematoxylin and eosin (H&E). The paraffin-embedded sections were also subjected to immunohistochemical staining with the anti-Cxcl5 antibody (15 μg/ml) and the anti-pro-SPC antibody (1 μg/ml) along with the peroxidase polymer anti-rabbit IgG reagents by using the Polymer method with ImmPRESS reagent (Vector Laboratories, Burlingame, CA, USA).

Staining was performed using standard procedures (ImmPRESS Reagent, Vector Laboratories, Burlingame, CA, USA, or TUNEL kits, R&D, Minneapolis, MN, USA).

Human tumor tissue array slides containing squamous cell carcinomas, adenocarcinoma, larger cell carcinoma, and matched normal lung tissues were obtained from US Biomax, Inc. (Rockville, MD). The immunohistochemistry procedure was performed by Biomax Inc. on two tissue microarray slides. As described previously [14] , [15] , tissue array slides were deparaffinised, hydrated and subjected to antigen retrieval. The slides were then incubated in 2.5% normal horse serum for 30 min at room temperature followed by incubation with the primary antibody (1∶100 dilution) for 1 h at room temperature. The affinity purified rabbit anti-Bit1 antibody (HPA012897) which was previously tested for its specificity [14] , [15] was purchased from Sigma. Rabbit normal serum was used as negative control antibody to replace the primary antibody on control slide with 1 hr incubation. Tissue array slides were then washed and incubated with ImmPRESS reagent (Vector Laboratories) followed by treatment with peroxidise substrate DAB solution (DAKO Cytomation). The slides were scored for average Bit1 staining intensity by two investigators with no knowledge of the pathologic status of the samples. The average staining was graded as 0, no staining; 1, slight staining; 2, moderate staining; and 3, strong staining.

For immunohistochemical analysis of TSG-6, rabbit polyclonal anti-TSG-6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:500 was used as the first antibody and incubated for 30 min, followed by ImmPRESS Reagent (VECTOR LABORATORIES, Burlingame, CA, USA) as the secondary antibody. Counterstaining was then performed before the sections were examined under a light microscope.

Formalin-fixed, paraffin-embedded serial sections were dewaxed, rehydrated, and subjected to heat-induced epitope retrieval using Target Retrieval Solutions, Citrate pH 6 or Tris/EDTA pH 9 (Dako, Agilent Technologies, Santa Clara, CA, USA), in an electric pressure-cooker for 20 min at 120 °C. To quench endogenous peroxidase activity, slides were incubated in 3% (v/v) hydrogen peroxide in methanol for 25 min. Nonspecific binding was blocked using 10% (v/v) fetal bovine serum in phosphate-buffered saline containing 0.1% Triton X100 for 1 h. Sections were incubated at 4 °C overnight with anti-human THPO (Antibodies-Online GmbH, Aachen, Germany), VEGF-A (Santa Cruz Biotech., Dallas, TX, USA), or Flt-1 (Santa Cruz Biotech.) rabbit polyclonal antibodies, or anti-human THPOR (R&D Systems, Minneapolis, MN, USA) or Flk-1 (Santa Cruz Biotech.) mouse monoclonal antibodies, or anti-human HIF-1α (Abcam, Cambridge, UK) rabbit monoclonal antibody. Negative controls were performed with nonimmune serum instead of the primary antibody. The slides were then stained with ImmPRESS Reagent (Vector Laboratories, Burlingame, CA, USA) for 30 min, revealed using the ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories), and counterstained with Mayer’s hematoxylin.