A technique for localizing LDL by immunofluorescence in formalin-fixed and paraffin-embedded atherosclerotic lesions

A protocol has been developed by which apo beta, the protein component of plasma low density lipoproteins (LDL), can be localized in four micron-thick sections of formalin-fixed and paraffin-embedded arteries with atherosclerotic involvement from man or non-human primate. An immunofluorescence procedure was employed that utilized affinity column-purified antibody coupled to fluorescein isothiocyanate (FITC). Although fixation reduced the intensity of fluorescence relative to that in cryostat sections, sufficient sensitivity was usually achieved when microscopy was performed using incident-light fluorescence together with a filter system adjusted for maximum absorption of FITC and by using oil immersion objectives. Furthermore, the use of various counterstains altered the color of the autofluorescence, normally strongly yellow, thus greatly enhancing the contrast and visualization of the apple green specific fluorescence. Non-chelated eriochrome black reduced tissue autofluorescence to barely visible levels, a feature useful in black and white photographs to demonstrate the localization pattern of LDL in arteries. This stain also reduced non-specific fluorescence. Other counterstains that proved useful were emthyl green, flazo orange and chelated eriochrome black. The latter two were particularly valuable for cellular detection. At low magnification LDL localization in atherosclerotic lesions from both human aortas and coronary arteries from monkeys fed an atherogenic diet, gave a pattern similar to those reported in cryostat sections of human lesions. At higher magnification, the improved detail of LDL localization could presumably be discerned as a result of immobilization of the soluble antigen by fixation, and by the ability to cut thinner sections of embedded material. This immunofluorescence procedure to localize LDL in formalin-fixed and paraffin-embedded atherosclerotic lesions should prove useful in both prospective as well as retrospective studies on clinical or chemical-pathological correlative studies on human tissues or in studies on atherogenesis using animal models.