Photomicrographs of sequential sections after skin biopsy from a patient with acute cutaneous GVHD: enzyme and antibody treatments. Representative adherence assay results of experimental manipulations in sequential sections. (A-E) Methyl green-thionin stain; original magnification × 40. Digestion of matrix elements was performed for 30 minutes at 37°C with the following: heparitinase 2 from Flavobacterium heparinum (Calbiochem, La Jolla, CA), 5 mU/mL in buffer (100 mM Na acetate,10 mM Ca acetate, pH 7), keratanase from Pseudomonas species (Calbiochem), 0.5 U/mL in buffer (50 mM Tris HCl, pH 8), hyaluronidase from Streptomyces hyalurolyticus (Sigma, St.Louis, MO), 20 U/mL in PBS buffer. For antibody treatments, assays were performed on alternating sequential sections in the presence of the function-blocking mAb Hermes-1 (10 μg/mL; gift from Dr Brenda Sandmaier, Fred Hutchinson Cancer Research Center) or rat IgG2a isotype antibody (control). Note the characteristic appearance of arcuate, palisading adherent lymphocytes (dark dots) binding to dermal papillary structures (as shown in panels A, B, and D). (A) Heparitinase 2 treatment of skin section. (B) No enzyme treatment; section incubated with PBS (matched control). (C) Hyaluronidase treatment of skin section. (D) Keratanase treatment of skin section. (E) Lymphocytes incubated with anti-CD44 mAb Hermes-1. Notice the absence of binding in the section treated with hyaluronidase (C) and in the section in which the assay was performed in the presence of Hermes-1 (E).