Alternate adjacent tissue sections from each animal were processed for single-labelling of ME utilizing either an immunoperoxidase or immunogold method. Sections were immersed in cryoprotectant solution (25% sucrose, 3% glycerol in 0.05 M PB) for 30 min and then briefly immersed in Freon followed by liquid nitrogen. This ‘freeze-thaw' method increases penetration of antibodies into the surface of the tissue with a minimal disruption of morphology6162. Tissue sections were incubated in a solution containing ME antibody for two nights at 4 °C. For visualization utilizing the avidin–biotin detection method63 sections were incubated with a biotinylated IgG for 30 min followed by 30 min in avidin–biotin complex solution (Vector Laboratories) and 2 min in a DAB-hydrogen peroxide solution. For visualization by immunogold, these sections were incubated with secondary antibody conjugated to gold particles. Sections were rinsed in citrate buffer and colloidal gold particles were enhanced by silver enhancement for 6 min 30 s using the IntenSEM kit (GE Healthcare Life Sciences, Pittsburgh, PA). All incubations, except the primary antibody incubation, were carried out at room temperature with continuous agitation on a shaker table and sections were rinsed between incubations in the appropriate buffer (either 0.1 M Tris-saline or 0.1 M PB). The primary antibody incubation buffer also contained 0.1% BSA.