Dual Immunofluorescence for Transporter Localization

Drug efflux transporter localization was identified using IHC as described previously (18 (link)). Dual immunofluorescence (IF) on frozen humanized mouse and NHP sections was performed on the Bond fully automated slide staining system (Leica Microsystems) using Bond Polymer Refine Detection kit (DS9800). Slides were allowed to sit at room temperature for 30 min and then fixed in 10% formalin for 15 min. They were then placed in Bond wash solution (AR9590). Antigen retrieval was done at 100°C in Bond epitope retrieval solution 2 (pH 9.0; AR9640) for 10 min. Staining was performed first using CD4 1F6 antibody (clone BC/1F6, Abcam) at 1:50 dilution for 1 hour with Bond polymer and post-primary reagents and Cy5 fluorochrome (PerkinElmer) for 15 min. Antigen retrieval was done at again 100°C in Bond epitope retrieval solution 2 (pH 9.0; AR9640) between protocols. Slides were then stained with CD3 (clone LN10, Leica) Ready-to-Use antibody for 15 min and Dako Envision mouse secondary for 30 min. Cy3 fluorochrome (PerkinElmer) was applied for 15 min. IF slides were counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA) and mounted with ProLong Gold antifade reagent (P36934, Life Technologies).