Anti ki67

Paraffin-embedded tissue blocks were sectioned into 3 μm slices and mounted on poly-L-lysine-coated slides. After deparaffinization, the slides were blocked by treatment with 3% hydrogen peroxide for 10 min and subjected to antigen retrieval by heating in 10 mM citrate (pH 6.0) for 15 min in a microwave. Next, anti-CD31 (1:100 dilution; BD) and anti-Ki67 (1:200 dilution; Novocastra, Newcastle upon Tyne, UK) antibodies were applied onto the sections and incubated overnight at 4 °C. Following repeated washings with PBS, the sections were treated with horseradish peroxidase/Fab polymer conjugate (Polymer detection system, Zymed, South San Francisco, CA, USA) for 30 min. After rinsing with PBS, the sections were incubated with peroxidase substrate diaminobenzidine (1:20 dilution, Zymed) for 5 min. Thereafter, the sections were counterstained with Gill’s hematoxylin for 2 s, dehydrated with serial ethyl alcohol, cleared with xylene, and mounted for analysis.

Immunostaining was performed on de-paraffined tissues sections after antigen retrieval (simmering in 0.1M citric acid, pH6.0, at 145°C for 10 minutes in a pressure cooker). The Vectastain Elite ABC system (Vector Laboratories, Burlingame, CA) was used following manufacturer’s instructions. The antibodies used were anti-pSTAT5 (9359, 1:500, Cell Signaling), anti-pSTAT3 (9145, 1:500, Cell Signaling), and anti-Ki67 (2011–11, 1:500, Novocastra), and were incubated at 4°C overnight. Apoptotic cells were determined by the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI). DAPI counterstain was used to visualize nuclei. For normal ducts and ADH-adjacent ducts, TUNEL-positive cells were scored in at least 10 high power (x40) fields per section, and at least 1000 cells were counted for each sample. All ADHs and UDHs were included for quantifying TUNEL-positive cells.

HE and immunohistochemistry were performed on formalin-fixed paraffin-embedded liver sections as described previously [46 (link)]. The following antibodies were used for staining: anti-α-SMA (Abcam, ab124964), anti-PCNA (Cell Signaling Technology, 2586), and anti-Ki67 (Novocastra, NCL-Ki67p).
To detect collagen deposition, paraffin sections were stained with Sirius red dissolved in picric acid solution according to the manufacturer’s recommendations.
For F4/80 immunofluorescence staining, liver cryosections were fixed in precooled acetone for 10 minutes and washed with phosphate-buffered saline (PBS) three times. After immersion in diluted normal goat serum, sections were sequentially incubated with anti-mouse F4/80 antigen PE (eBioscience, 12–4801) overnight at 4°C. Nuclei were stained with DAPI for 5 minutes after two washes with PBS.
TUNEL assays were performed using an Apoptosis DNA Fragmentation Assay Kit (Clontech, #630107) to detect apoptotic cells. All images were visualized using a U-RFL-T microscope (Olympus, Tokyo, Japan). The positive cells or areas were counted or measured in at least five fields on each slide using ImageJ (NIH) software.

Histological and immunohistochemical analysis of lung tumors was performed as previously described [51 (link)]. Briefly, 2 µm sections from at least 3 different planes of the lung were cut and stained with H&E. Sections were scanned using a Mirax slide scanner and lung/tumor areas automatically scored by an algorithm programmed and executed using the Definiens software suite and visually controlled in a blinded way. Immunohistochemistry staining was done using an automatic staining machine (Leica Bond3) or manually processed. Sections were dehydrated and antigenic epitopes retrieved using a 10-mM citrate buffer and microwaving for 10 min Specimens were then incubated with anti-Foxp3 (eBioscience, 13-5773), anti-CD3ε (Santa Cruz, 101442), anti-Ki67 (Novocastra), anti-CD31 (Abcam, ab28364), and anti-cleaved Caspase 3 (Cell signaling, 9661). Primary antibody staining was detected by peroxidase-conjugated anti-rabbit IgG. Positive cells were counted on 20 randomly chosen tumor areas at ×400 magnifications in a double-blinded fashion. Images were captured with a Zeiss AxioImager Z1. Quantitative analysis was performed using HistoQuestTm software (TissueGnostics GmbH, Vienna, Austria, www.tissuegnostics.com). These experiments were independently replicated, with biological and technical replicates as detailed in the legend of the corresponding figure, with similar results.