Ab37056

Mouse colons were embedded in OCT after euthanasia. The tissues were sectioned at 4 μm thickness, and fixed with 4% formaldehyde for 30 min at RT. After washing with PBS, the section was blocked with blocking buffer (Zymed Laboratories Inc., San Diego, USA) before incubated with anti-Citrobacter antibody (Abcam, ab37056) overnight at 4°C as previously described[41 (link)]. The section was then stained with Secondary Fluorescent Antibody. After washing in PBS, the tissues were counterstained with mounting medium containing DAPI.

For immunohistochemical (IHC) stainings, distal colonic tissues from mice infected with C. rodentium were fixed in buffered formalin (Roti-Histofix; Carl Roth) at 4 °C for 24 h, dehydrated, and embedded in liquid paraffin. 3-µm sections were cut using a microtome (Leica) and processed for IHC applying the Tyramide Signal Amplification (TSA) Cy3 system (Perkin Elmer) according to the manufacturer’s protocol. To analyze the expression level of IRF-1, a primary antibody from Cell Signaling (D5E4) was used (1:50 dilution). To visualize the colonization of the mucosal surface with C. rodentium, a primary antibody from Abcam (ab37056, 1:1000) was applied. Both primary antibodies were used in combination with a goat-anti-rabbit biotinylated secondary antibody (Jackson Immuno Research). Epithelial cells were stained with Alexa Fluor 488 anti-mouse CD326 (Ep-CAM; G8.8; BioLegend, 1:100). Nuclei were counterstained with DAPI (Invitrogen). Pictures were acquired on a Leica TCS SP5 II confocal microscope using Leica LAS AF version 2.7.3.9723 software.

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).