Dab chromogen

Intestines were flushed with PBS, fixed overnight in 4% PFA at 4°C, dehydrated in 70% EtOH over night at 4°C, then embedded in paraffin using the swiss roll technique³⁴ . Immunohistochemistry and hematoxylin and eosin staining were performed by the Texas Medical Center- Digestive Diseases Center, Cellular Morphology core at Baylor College of Medicine. Briefly, sections were deparaffinized and subjected to antigen retrieval with Rodent Decloaker (Cat. #RD913, Biocare). The sections were then incubated with 3% hydrogen peroxide, followed by incubation in normal serum to block nonspecific protein binding. Sections were incubated 1 hour at room temperature with anti-Ki67 (1:75, Cat. #CRM325, Biocare); and anti-Olfm4 (1:500, Cat. #39141S, Cell Signaling). All antibodies were then detected with a Rabbit-on-Rodent HRP-Polymer (Cat. #RMR622H, Biocare) and visualized with DAB chromogen (Cat. #DB801, Biocare). All slides were counterstained with hematoxylin, dehydrated, and mounted with a permanent mounting medium. A Nikon Ci-L bright field microscope was used for imaging at the Integrated Microscopy Core (Baylor College of Medicine). All images quantified were taken at 20X magnification and quantified using ImageJ24 (https://imagej.nih.gov/ij/). Detailed methods for crypt and villus area quantification are included in the supplemental methods.

Immunohistochemistry was performed on tumor specimens obtained with informed consent from the brain tumor bank with Institutional Review Board approval. The tissue was paraffin-embedded shortly after operative resection, sectioned via microtome, and mounted. Prior to antigen retrieval, sections were deparaffinized in a rice steamer using deionized (DI) water for 20 min. The sections then underwent background block for 10 min (Cell Marque^®), a DI water washing, and peroxide block for 10 min (Cell Marque^®), prior to incubation overnight at 4 °C with either the iC3b neoantigen monoclonal antibody (Quidel^®) at 1:4,000 (0.25 μg/mL) or mouse IgG control (Sigma^®) at 1:800 (0.25 μg/mL). After washing with PBS, the sections were incubated with HiDef amplifier (Cell Marque^®) for 10 min, washed again with PBS, and incubated for another 10 min with HiDef HRB polymer (Cell Marque^®). After another PBS wash, the sections were developed with DAB Chromogen (Biocare^®), washed with DI water, and finally were counterstained with immunohematoxylin (American Master Tech^®).