A simple reliable method for producing electron dense markers of uniform size for use in immunoelectron microscopy

A method is presented for the preparation of electron dense markers of defined size for use in immunoelectron microscopy, The electron dense markers were formed by the covalent binding of rabbit IgG and horse spleen ferritin mediated by glutaraldehyde. We demonstrated that the conjugate size, i.e. the number of ferritin grains per unit conjugate, was directly related to the time of incubation at 4 degrees C in a 1000 molar excess of glutaraldehyde. Conjugates were separated on the basis of molecular size by Sepharose 4B column chromatography. Three different sized conjugates were used to delineate pseudomonas toxin receptors on the surface of mouse LM fibroblasts; similar numbers of receptors were observed with all conjugates.