Goat anti rabbit antibody conjugated to polymer horseradish peroxidase

After deparaffinization, antigen retrieval was performed by heating microwave (700 W) for 20 min in a 10 mM citrate buffer at pH 6.0, with a cool down period of 20 min afterwards. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 20 min. Slides were than incubated with the primary anti-human-PSMA mouse monoclonal antibody, YPSMA-1 (Abcam, Cambridge, UK), diluted at 1:400 in 1% bovine serum albumin/phosphate-buffered saline (1% BSA/PBS) for 1 h at room temperature. The secondary step consisted of incubation with rabbit anti-mouse antibody conjugated to polymer-horseradish peroxidase (DAKO, Glostrup, Denmark), diluted at 1:100 in 1% BSA/PBS with 1% AB serum. For the tertiary step, goat anti-rabbit antibody conjugated to polymer-horseradish peroxidase (DAKO, Glostrup, Denmark) was used, diluted at 1:100 in 1% BSA/PBS with 1% AB serum. Both the secondary and tertiary step required incubation for 30 min at room temperature. Next, the slides were immersed for 10 min in a solution of 0.05% 3,3′-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) and 0.03% hydrogen peroxide in PBS for the visualization of the signal as brown staining. After washing with demineralized water, the slides were slightly counterstained with hematoxylin, dehydrated and mounted with Eukitt mounting medium (Sigma-Aldrich, Steinheim, Germany).

After deparaffinization, antigen retrieval was performed by heating microwave (700 W) for 20 min in 0.1 M Tris/HCl buffer at pH 9.0, with a cool down period of 20 min afterwards. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in Tris-buffered saline (TBS) for 20 min. Slides were then incubated with a normal goat serum diluted at 1:10 in TBS for 30 min at room temperature. Afterwards, the slides were incubated with the primary anti-human-GRPR rabbit polyclonal antibody, ab39963 (Abcam, Cambridge, UK), diluted at 1:250 in 1% BSA/TBS overnight at 4 °C. Only a secondary step with goat anti-rabbit antibody conjugated to polymer-horseradish peroxidase (DAKO, Glostrup, Denmark) was applied, diluted at 1:100 in 1% BSA/TBS with 1% AB serum for 60 min at room temperature. Next, the slides were immersed for 10 min in a solution of 0.05% 3,3′-diaminobenzidine (Sigma-Aldrich, Steinheim, Germany) and 0.03% hydrogen peroxide in PBS for the visualization of the signal as brown staining. After washing with demineralized water, the slides were slightly counterstained with hematoxylin, dehydrated and mounted with Eukitt mounting medium (Sigma-Aldrich, Steinheim, Germany).

After deparaffinization, microwave antigen retrieval (700 W) was performed for 20 min in 10 mM Tris/1 mM EDTA buffer at pH 9.0, with a cool down period of 20 min afterwards. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in PBS for 20 min. Slides were incubated with a normal goat serum diluted at 1:10 in PBS for 30 min at room temperature. The primary step consisted of incubation with rabbit anti-human antibody VEGF A-20 sc-152, (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted at 1:200 in 1% BSA/PBS for 1 h at room temperature. Only a secondary step with goat anti-rabbit antibody conjugated to polymer-horseradish peroxidase (DAKO, Glostrup, Denmark) was applied, diluted at 1:100 in 1% BSA/TBS with 1% AB serum for 30 min at room temperature. Next, the slides were immersed for 10 min in a solution of 0.05% 3,3′-diaminobenzidine (Sigma-Aldrich, Steinheim, Germany) and 0.03% hydrogen peroxide in PBS for the visualization of the signal as brown staining. After washing with demineralized water, the slides were slightly counterstained with hematoxylin, dehydrated and mounted with Eukitt mounting medium (Sigma-Aldrich, Steinheim, Germany).