Immunohistochemical localization of heparan sulfate-containing proteoglycan in normal human skin with monoclonal antibodies: comparison with that of fibronectin

Heparan sulfate-containing proteoglycan (HSPG) has been immunohistochemically demonstrated in the basement membrane zones (BMZs) in the human skin [1, 6, 8, 11]. We reported on the isolation of an unique HSPG, the core protein of which has an affinity for fibronectin (FN), from human placenta and the preparation ofmonoclonal antibodies (MoAbs), HS42 and HS47, which recognize the core protein [8]. In the present paper we studied the detailed localization of the antigen reacting with these MoAbs in the human skin and compared it with that of FN. Normal skin specimens were embedded in OCT compound (Miles Laboratories Inc., Naperville, IL, USA) and quickly frozen in liquid nitrogen with and without preincubation in phosphate-buffered saline (PBS) containing 10% glycerol for 1 h at 4~ for immunoperoxidase and immunofluorescent stainings, respectively. For immunofluorescent microscopy, the cryostat sections were stained with rabbit antiserum to FN as described previously [12] and rhodamine conjugated goat rabbit-specific IgG (Tago Inc., Burlingame, CA, USA). After rinsing, the same sections were then stained with the MoAb and FITC conjugated rabbitmouse-specific IgG (DAKOPATTS a/s, Glostrup, Denmark). The control sections were incubated with PBS instead of the MoAbs. Immunofluorescences of both FITC and rhodamine on the same area of each section were photographed.