Dakocytomation lsab2 system hrp kit

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. The antigen was retrieved with 0.01 M citrate buffer (pH 6.0) by heating the sample in microwave for 10 min. The tissue sections were then placed in 3% hydrogen peroxide for 5 min to inactivate the endogenous peroxidase, and blocked for 30 min with normal horse serum (DakoCytomation LSAB2 System-HRP kit; DakoCytomation, Glostrup, Denmark). The primary antibodies used for this study were Ki-67 rabbit polyclonal antibody (1:300; Abcam, Cambridge, MA, USA), VEGF and SIRT1 mouse polyclonal antibodies (1:150; all from Abcam), cleaved caspase-3 rabbit polyclonal antibody (1:300; Cell Signaling), and B-cell lymphoma-extra large (Bcl-xL) mouse monoclonal antibodies (1:100: Santa Cruz biotechnology, Dallas, TX, USA). The prediluted primary antibodies were applied overnight at 4 °C. The slides were then treated with biotinylated secondary antibody for 30 min at room temperature, followed by the treatment with streptavidin-HRP and 3,3′-diaminobenzidine solution for another 10 min at room temperature. Tissue sections were counterstained with hematoxylin.

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. The antigen was retrieved with 0.01 M citrate buffer (pH 6.0) by heating the sample in an autoclave (CHS-ACCE-860, JW Pharmaceutical, Seoul, Korea) at a controlled final temperature of 121°C for 5 min. The tissue sections were then placed in 3% hydrogen peroxide for 5 min to inactivate the endogenous peroxidase, blocked for 10 min with normal horse serum (DakoCytomation LSAB2 System- HRP kit; DakoCytomation, Glostrup, Denmark), and incubated with the primary antibodies against β-catenin, survivin, E-cadherin, and Snail overnight at 4°C. The slides were then treated with the biotinylated secondary antibody for 30 min at room temperature, followed by streptavidin-HRP and 3, 3′-diaminobenzidine solution for another 10 min at room temperature.