Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody.

Abstract

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8 was employed. In neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37.degree.-40.degree. C. Unfixed cryostat tissue sections were reacted with this antibody at successively higher temperatures. Denaturation was operationally defined as the point at which type IV-specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50.degree.-55.degree. C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60.degree. C, the highest temperature at which this technique can be used confidently. The stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution. Conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.