Ultrastructural Localization of PMP70 in Mouse Retina

A mouse eye was prepared and pre-embedding labelled performed using an antibody against the 70-kDa peroxisomal membrane protein, PMP70, before embedding for TEM as described [10 (link)]. In brief, a mouse eye sacrificed at 1.5 hours after light onset was fixed in 4% paraformaldehyde. Sections were cut using a cryostat and permeabilised using 0.02% saponin in 1% BSA in PBS for 30 mins. Antibody labelling was performed using an antibody against PMP70 (Novus Biologicals) in 1% BSA in PBS overnight at 4°C. Secondary Nanogold (Nanoprobes) in 1% BSA in PBS was applied to the sections for 2 hrs before re-fixing in 2% PFA, 2% glutaraldehyde in 0.1M cacodylate buffer for 1 hr. Gold enhancement solution (Nanoprobes) was applied to the sections following the manufacturers guidelines on ice at 4°C before preparing for TEM as described below.