Bond er2 buffer

Serial sections (5 microns) of paraformaldehyde-fixed paraffin-embedded samples underwent antigen retrieval using Leica Bond ER2 Buffer (Leica Biosystems) for 20 min at 100°C before staining with 1μg/ml Desmin Rabbit polyclonal antibody (Abcam cat#ab8592) and 1μg/ml pStat3 (clone D3A7, Cell Signaling) for 1 hour using Leica Protocol F (Molecular Imaging Core facility, MSKCC). Quantification of Desmin/pStat3 staining was performed using ImageJ/FIJI (NIH). At least 19 fields at 400X were randomly selected and evaluated. The results were expressed as percentage of immunostained cells/over total area of tissue. To discriminate between cancer and stromal cells fortified H&E staining was also performed (HistoServ Inc, Germantown, MD). A color deconvolution algorithm was then used, with RGB vectors for the stromal component and counterstain/background stain created from ROIs drawn from example images (Molecular Imaging Core facility, MSKCC). Appropriate thresholds were then set for each cell type of interest and area measurements were taken for all images. To rule out possible non-CAFs/non-cancer cells component, specific staining for CAFs (desmin -murine CAFs) and cancer cells (human pankeratin) was also performed in serial section slides. Stroma-tumor niches were evaluated as area of tissue slide with the co-presence of pankeratin positive cells and stroma cells.

The carcasses were fixed in 4% paraformaldehyde and processed for paraffin embedding using Leica ASP6025 tissue processor (Leica Biosystems, Buffalo Grove, IL). Freshly cut 5 micron paraffin sections of the peritoneal cavity were stained for sequential double immunofluorescence on Leica Bond RX (Leica Biosystems, Buffalo Grove, IL) with 0.6 ug/ml CD3 Rabbit Polyclonal (Dako cat#A0452, Santa Clara, CA) for 1 hour and using 10 min of 1:200 Tyramide Alexa Fluor488 detection (Life Technologies, Gaithersburg, MD) on Protocol F, followed by 4 ug/ml hCEA Rabbit Monoclonal (Abcam cat#15987, Cambridge, MA) for 1 hour and using 10 min of 1:200 Tyramide Alexa Fluor568 detection (Life Technologies, Gaithersburg, MD) on Protocol F. The sections were pre-treated with Leica Bond ER2 Buffer (Leica Biosystems, Buffalo Grove, IL) for 20 min at 100°C before each staining. After staining the sections were dehydrated and mounted with Permount for digital scanning with Pannoramic Confocal (3DHistech, Budapest, Hungary) using 40X water objective.

MYC IHC was performed at the Molecular Cytogenetics Core Facility. Paraffin sections with 5-μm thickness were stained for IHC on Leica Bond RX (Leica Biosystems) with 8 μg ml⁻¹ c-myc rabbit monoclonal antibody (Cell Signaling Technologies,13987) for 1 h on the basis of the default manufacturer Protocol F. The sections were pretreated with Leica Bond ER2 buffer (Leica Biosystems) for 20 min at 100 °C before each staining. After staining the sections were dehydrated and mounted with Permount for digital scanning with Pannoramic Confocal (3dHistech) using ×40 water objective.