Ultra vision lp detection system

After animal sacrifice, the brain was soaked in formalin after dehydration and 3-μm-thick microtome TMA slides were embedded in paraffin wax. The slides were individually immunostained with anti-ANP antibodies, employing the Ultra Vision LP Detection System (Vector Laboratories, Waltham, MA, USA). The embedded tissue was kept overnight at 60 °C for the pull-out embedded paraffin. After overnight rehydration with serious xylene and graded ethanol, “H₂O₂” blocking buffer was used to block peroxidation on embedded tissue and then washed with “ddH₂O”. The slides were treated with citrate buffer by heating with a microwave for the required time interval after cooling at room temperature. These slides were incubated with primary antibody for 2 h (1:100) after blocking with blocking buffer. Thereafter, the slides were incubated with the universal secondary antibodies for 30 min before staining with hematoxylin, and the slides were stained with chromogen for 5 min. Finally, slides were counterstained with hematoxylin, dehydrated by a series of graded ethanol to xylene washes, and fixed on coverslips with mounting media (Sigma Chemical, Saint Louis, MO, USA). Mounted tissue sections were viewed under a microscope (magnification 200×).

The slides were immunostained with anti-p-PKCδ T505/507 antibodies (1:100 dilution; bs-208 3727R; Bioss) using the Ultra Vision LP Detection System (Vector Laboratories, California, USA) according to the manufacturer’s instructions. The tissue sections were deparaffinized and rehydrated. After three washes in PBS, hydrogen peroxide blocking buffer was used to block endogenous peroxidase activity for 10 min. Ultra V Block was incubated for 5 min at room temperature to block nonspecific background staining. Then, the sections were incubated with a primary antibody against p-PKCδ T505/507 (1:100 dilutions; bs-3727R; Bioss) at 4 °C overnight in a moist chamber. After incubation with horseradish peroxidase for 20 min at 37 °C, these antibodies were located using a universal secondary antibody formulation conjugated to an enzyme-labeled HRP polymer. After staining with an appropriate substrate/chromogen for 5 min, the slide was counterstained with Harris hematoxylin, and cover slipped with permanent mounting media (Sigma Chemical, Missouri, USA). The expression of p-PKCδ was then detected using microscopy (Olympus, Tokyo Japan).