Anti ly 6g

Tumours were embedded in Tissue-Tek O.C.T. media (Sakura) on dry ice and immediately stored at −80 °C until sectioning. 10 μm thick sections were collected on Leica cryostat and stored at −80 °C until staining. Slides were removed from −80 °C, fixed in cold acetone or acetone:methanol. Following washing in PBS, slides were incubated with protein block and subsequently incubated with specific antibodies overnight. Antibodies used: anti-CD4 (553043; BD Bioscience), anti-CD8 (553027; BD Bioscience), anti-MHCI (15681; Abcam), anti-FoxP3 (54501; Abcam), anti- Ly-6G (MAB1037; R&D System), anti-CD68 (53444; Abcam). Appropriate horseradish peroxidase (HRP) conjugated secondary antibodies were used for detection of the primaries and developed with DAB chromogen. Purified Rat IgG2a, k for the CD8a and CD4 (553027; BD Pharm); clone RTK2758 for the MHCI (70636; BioLegend); for Ly6G (400601; Biolegend); for FoxP3 (I-1000; Vector) were used as negative controls. Slides were counter stained with haematoxylin and eosin (H&E) and dehydrated in ethanol and xylene. Slides were then cover slipped and imaged with an Aperio ScanScope at 20x magnification.

For histology, tissues were fixed in 10% neutral buffered formalin or Carnoy’s fixative (60% methanol, 30% chloroform, and 10% acetic acid) before paraffin embedding. Images were obtained with an Eclipse i80 microscope (Nikon Instruments, Melville, NY, USA). Paraffin-embedded 5-μm-thick sections were stained with H&E for gross morphology and with Alcian blue to detect goblet cells. Immunohistochemistry and immunocytochemistry were performed with appropriate antibodies on paraffin-embedded sections [18 (link), 21 (link)]. Antibodies used were rabbit anti-DCLK1 (1:200, ab31704) and Anti-CR (1:250, ab37056) from Abcam, Cambridge, UK; mouse anti-Notch1 NICD (1:200, clone OTI3E12) from Origene, Rockville, MD; rabbit anti-Hes1 (1:200, #11988) and mouse anti-Ki-67 (1:200, #9449) from Cell Signaling Technology, Danvers, MA; anti-CD68 (1:200, NB600-985) from Novus Biologicals (Littleton, CO, USA), Anti-Muc2 and anti-NE (1:200) Santa Cruz, Dallas, TX; Anti-CD11c and anti-F4/80 (Thermo Fisher), and anti-Ly6G (R&D, Minneapolis, MN). Antibody controls included omission of the primary antibody or detection of endogenous IgG staining with goat anti-mouse or anti-rabbit IgG (Calbiochem, San Diego, CA, USA).