Lasb kit

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 above^{79 (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 primary antibodies for Ki67 (1:500) (Abcam, Cambridge, MA), green fluorescent protein (GFP) and β-catenin (Cell Signaling Technology, Danvers, MA), followed by secondary antibodies and DAPI (1:1000) (Vector Laboratories, Burlington, ON). 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. Quantification was performed by three blinded investigators.

Cells were fixed using 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. After blocking with 1% BSA, cells were incubated overnight at 4 °C with a primary antibody (1:500 dilution) for cell proliferation Ki67 (ab15580, Abcam, Cambridge, MA). Cells were then incubated with secondary antibodies (1:1000 dilution, A-11034, Invitrogen, Carlsbad, CA, USA) and DAPI (Vector Laboratories, Burlington, ON, Canada) for nuclei at room temperature for 2 h. The image was captured using a Nikon TE-2000 digital microscope equipped with a Hamamatsu C4742-80-12AG camera. The number of antibody-labeled cells was quantified from five separate images by three blinded investigators.
Similarly, sections of terminal ileum were immunostained for Ki67 (ab15580, Abcam, Cambridge, MA) using the same protocol as above. For subsequent reactions, a streptavidin–biotin complex peroxidase kit (LASB + Kit, Dako, Denmark) was used and slides analyzed the same way as mentioned above.