Bond polymer refine detection kit ds9800

Slides were covered with covertiles (Bond Universal Covertiles, Leica biosystems) and baked for 10 mins at 57°C. Slides were deparaffinizied in dewax solution and rehydrated in decreasing concentrations of EtOH. Tissue sections were then incubated in Ag retrieval solution (pH 6 or 9) at 95°C for 20 mins. Tissue sections were incubated in 3% hydrogen peroxide (Bond Polymer Refine Detection Kit DS9800, Leica Biosystems) for 15 mins to block endogenous peroxidases. Next, tissue sections were incubated in serum-free protein block solution (Dako X0909) for 30 mins to block nonspecific antibody binding. After the first staining cycle, Fab fragments (AffiniPure Fab Fragment Donkey anti-mouse (715–007-003) or anti-rabbit IgG (711–007-003)) against that primary antibody species were used to block carryover staining whenever there was a repeat of same primary antibody species. Primary antibody staining was performed for 1 hour at room temperature followed by secondary antibody staining. Polymer detection system (Bond Polymer Refine Detection Kit #DS9800, Leica Biosystems) was used for horseradish peroxidase signal amplification. Chromogenic revelation was performed using ImmPact AEC (3-amiino99-ethylcarbazole) substrate (Vector Laboratories, SK4205) for preset incubation times. Slides were counterstained with hematoxylin (Bond Polymer Refine Detection Kit, DS9800, Leica Biosystems.