Biotin conjugated mouse secondary antibody

Immunohistochemistry was performed in the Georgetown University Medical Center Histopathology and Tissue Shared Resource. Five micron sections from formalin fixed paraffin embedded tissues were de-paraffinized with xylene and rehydrated through a graded alcohol series. Heat induced epitope retrieval (HIER) was performed by immersing the tissue sections at 98°C for 20 minutes in Target retrieval solution, high pH (Dako). Immunohistochemical staining was performed using the VectaStain Kit from Vector Labs according to manufacturer’s instructions. Briefly, slides were treated with 3% hydrogen peroxide for 10 minutes. Endogenous biotin was blocked using an avidin/biotin blocking kit from Invitrogen. The slides were then treated with 10% normal goat serum for 10 minutes and exposed to primary antibody for STAG2 (1:50, Santa Cruz, sc81852) for 1 hour at room temperature. Slides were then exposed to biotin-conjugated mouse secondary antibody (Vector Labs), Vectastain ABC reagent and DAB chromagen (Dako). Slides were counterstained with hematoxylin (Fisher, Harris Modified Hematoxylin) at a 1:8 dilution for 2 minutes at room temperature, blued in 1% ammonium hydroxide for 1 minute at room temperature, dehydrated, and mounted with Acrymount.

To determine the expression of ACE-liposome targeted cortactin plasmids in mouse lung tissue, paraffin blocks of lung tissues were prepared and 10 μm microscope slides obtained. Serial sections of each specimen were deparaffinized and rehydrated in serial graded ethanol. Antigens were retrieved with 50 mM citrate buffer, pH 6.4 and by heating slides in a 98°C water bath, for 15 min. Endogenous peroxidase activity was blocked in methanol containing 3% hydrogen peroxide and endogenous biotin was blocked with avidin biotin block. After blocking with an IgG blocker and normal horse serum, the sections were incubated with a mouse monoclonal antibody to mouse c-myc (9E10, Sigma Chemical Company, St Louis, MO, USA) 1: 500 dilution for 1 hour, followed by 30 min incubation with a biotin conjugated mouse secondary antibody (Vector Laboratories Inc., Burlingame, CA, USA). The ABC complex and 3,3′-diaminobenzidine (Vector Laboratories, Inc., Burlingame, CA) solutions were used serially, and the slides were counterstained with hematoxylin.