Sequential use of the PAP and immunogold staining method for the light microscopical double staining of tissue...

Double immunoperoxidase staining using different couplers can give various combinations of colours on a single tissue section to achieve a comparable picture of different antigens. However, the colour combinations achieved to date are not entirely satisfactory. A double immunostaining procedure is introduced here, combining the peroxidase anti-peroxidase (PAP) and immunogold staining (IGS) methods. The IGS method is a new, simple, sensitive and reliable approach to immunostaining at the light microscopic level. It was carried out in three ways. Firstly, a two-step method was used in which the second layer was goat anti-rabbit IgG absorbed onto gold particles (GAR/Au20). Secondly, a three-step method was employed where the second layer was unlabelled goat anti-rabbit IgG and the third layer was a rabbit antibody to peroxidase absorbed onto the gold particles (RAP/Au20) and acting as a gold-labelled IgG antigen. The third method combined the first two methods using GAR/Au20 as th second layer and RAP/Au20 as the third layer which increased the amount of bound gold and enhanced the red colour, providing a better picture. The use of gold-labelled antibodies in double immunostaining has great potential value for many studies including that of the diffuse neuroendocrine system of the gut.