Immunohistochemistry of Mucosal α4β7 Expression in IBD Patients

Cryosections from colonic biopsies of IBD patients were used for immunohistochemistry. Tissue sections were fixed in 4% paraformaldehyde, followed by sequential incubation with avidin/biotin- (Vector Laboratories), and protein-blocking reagent (Roth) to suppress unspecific background staining. Sections were incubated with primary antibodies specific for human CD3 (rat anti-human CD3, Bio-Rad, Cat. MCA1477), human CD4 (rat anti-human CD4, Bio-Rad, Cat. MCA484G), and FITC-labeled vedolizumab (Entyvio^®, Takeda). Furthermore, sections were incubated with an isotype matched control antibody as negative controls. Subsequently, samples were incubated with Alexa 647 conjugated secondary antibodies. Nuclei were counterstained with DAPI before final analysis by confocal microscopy (Leica SP8 Microscope). Positive cells in 6–10 high power fields (HPFs) were subsequently counted in all patients. In some images, an inset of a higher magnification was included to better identify the stained nuclei.