The detection of mast cell subpopulations in formalin-fixed human tissues using a new monoclonal antibody specific for chymase.
Chymase is an important marker for human mast cells as well as a mediator of inflammation and matrix remodelling, but research into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue. A monoclonal antibody to chymase (designated CC1) was prepared by immunizing a mouse with chymase purified from human skin, fusing the splenocytes with NS-1 myeloma cells, and screening the hybridoma supernatants by ELISA with recombinant human prochymase isolated from a baculovirus expression system. This antibody bound to chymase in western blots and bound selectively to cells with the morphology and distribution of mast cells in paraffin wax sections of skin, synovium, lung, and heart. In sequential sections and with double-labelling experiments, chymase was localized to cells which contained mast cell tryptase; in contrast to previous reports, no evidence was found for its presence in endothelial cells or any other cell type. The antibody permitted chymase-containing mast cells to be detected in formalin-fixed tissues, and the numbers identified were similar to those in tissues fixed with Carnoy's or ethanol fixatives. Immunocytochemistry with antibody CC1 provides for the first time a sensitive and specific means for the detection of chymase in routinely fixed tissues and should prove valuable in studying mast cell subsets in disease.
Buckley
M G
MG
Immunopharmacology Group, University of Southampton, Southampton General Hospital, Southampton, U.K.
McEuen
A R
AR
Walls
A F
AF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002452:Cell Count; D053818:Chymases; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D008168:Lung / Q000473:pathology; D008407:Mast Cells / Q000201:enzymology / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009206:Myocardium / Q000473:pathology; D012697:Serine Endopeptidases / Q000032:analysis*; D012867:Skin / Q000473:pathology; D013583:Synovial Membrane / Q000473:pathology; D016707:Tissue Fixation
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D005557:Formaldehyde; D012697:Serine Endopeptidases; D053818:Chymases