After sacrificing the pups on P9, 1 cm samples of terminal ileal tissue were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, cross-sectioned (5 μm), and counterstained with hematoxylin and eosin by standard protocols. Histology slides were blindly assessed by three independent investigators using a published scoring system in which confirmed NEC is defined as mice with a grade 2 or above79 (link).
Sections of terminal ileum were immunostained with 1 in 500 dilutions of primary antibodies for polyclonal rabbit anti-CBS antibody (Proteintech, 14787-1-AP) or pimonidazole (Hypoxyprobe) followed by incubation with 1 in 1000 diluted Alexa Fluor-conjugated secondary antibody (Invitrogen, Carlsbad, California, United States) and DAPI (Vector Laboratories, Burlington, ON) for visualization of cell nuclei. For subsequent reactions, a streptavidin-biotin complex peroxidase kit (LASB + Kit, Dako, Denmark) was used. Slides were analyzed using a Nikon TE-2000 digital microscope equipped with a Hamamatsu C4742-80-12AG camera. Three blinded investigators counted the number of antibody-labeled cells from a minimum of five images. Immunofluorescent images were quantified using ImageJ.
Sections of terminal ileum were immunostained with 1 in 500 dilutions of primary antibodies for polyclonal rabbit anti-CBS antibody (Proteintech, 14787-1-AP) or pimonidazole (Hypoxyprobe) followed by incubation with 1 in 1000 diluted Alexa Fluor-conjugated secondary antibody (Invitrogen, Carlsbad, California, United States) and DAPI (Vector Laboratories, Burlington, ON) for visualization of cell nuclei. For subsequent reactions, a streptavidin-biotin complex peroxidase kit (LASB + Kit, Dako, Denmark) was used. Slides were analyzed using a Nikon TE-2000 digital microscope equipped with a Hamamatsu C4742-80-12AG camera. Three blinded investigators counted the number of antibody-labeled cells from a minimum of five images. Immunofluorescent images were quantified using ImageJ.
Free full text: Click here
