Sequential immunostaining of GI biopsies. (A) Representative example of a GI biopsy stained sequentially for RARα, IL-23R, and T-bet using VIP chromogen and hematoxylin counterstain; positive cells stained purple, and CD8 stained brown (using DAB chromogen). Subcrypt regions are indicated by a white outline. Cells with high and low or no expression of individual markers are indicated with red and black arrows, respectively. Scale bars, 25 µm. (B) Sequential immunostaining was validated by demonstrating a close correlation between mononuclear cell numbers in sequentially stained sections enumerated using manual counting. Mononuclear cell numbers in the same sections were stained by using conventional single-stain IHC and enumerated by using automated cell counting with the Ariol system. P and r values are for Pearson correlation coefficients.