Stronger staining and higher sensitivity was observed when use ImmunoHistoProbe® Plus High Sensitivity Double Staining Polymer IHC Detection System (Pictures B, D, F) comparing with regular one step polymer detection system (Pictures A, C, E).
Archival formalin-fixed paraffin embedded human tissue sections were deparaffinized in xylene with 2 changes for 5 minutes each. Rehydrate through graded alcohol (100%, 95% and 70%). 1mM EDTA pH9.0 HIER 15’ or none. New ImmunoHistoProbe® Plus High Sensitivity Double Staining Polymer IHC Detection System was incubated @RT 15min+15min: Amplifier 15min, wash 3 x 2 min then add HRP/AP Polymer Detector for another 15 min. (Picture B, D, F). HRP Polymer Goat anti-Rabbit or mouse and AP Polymer Goat anti-Mouse or rabbit (1:1 mixture) was incubated @RT 20 min (Picture A, C, E). DAB Chromogen and AP-Red Chromogen. Hematoxylin counter staining. Serial tissue sections were used in order to compare staining intensity side by side.
A and B: Esophageal Carcinoma. Mouse anti-P53 (1:20,000) and Rabbit anti-CD146 (1:1000) cocktail, RT, 2 hr.
C and D: Tonsil, Mouse anti-Ki67 (1:10,000) and Rabbit anti-CD3 cocktail, RT, 2 hrs.
E and F: Colon Cancer, No HIER. Mouse anti-Smooth Muscle Actin (1:5000) and Rabbit anti-CEA (1:2000) cocktail, RT, 2 hrs.