Ultravision hydrogen peroxide block

Human colorectal cancer tissue sections were de-paraffinized in xylene and re-hydrated in a series of graded alcohols. After rinsing twice with PBS, the sections were then quenched the activity of endogenous peroxidase with Ultravision Hydrogen Peroxide Block (Thermo scientific, Barrington, IL) for 10 min and incubated with Ultravision Protein Block (Thermo scientific, Barrington, IL) for 10 min to reduce non-specific binding. Sections were incubated with an rabbit anti-B4GALNT3 polyclonal antibody (Sigma, St. Louis, MO, 1:100) diluted with 1% bovine serum albumin (MDBio, Inc., Taipei, Taiwan)/PBS for 16 h at 4 °C. After rinsing twice with PBS, the sections were processed using the Ultravision Quanta Detection System (Thermo scientific, Barrington, IL). Specific immuno-staining was visualized with DAB Quanto (Thermo scientific, Barrington, IL). All sections were counterstained with hematoxylin for 1 min and mounted with UltraKitt (J.T. Baker, Deventer, Holland). Negative controls were performed by replacing primary antibodies with rabbit IgG (SouthernBiotech, Birmingham, AL) at the same concentration.

For immunohistochemical analysis, formalin-fixed paraffin-embedded blocks of the mice’s liver tissue were cut into 2-µm sections. After deparaffinization and rehydration, sections were heated in a citrate buffer (10 mM, pH 6, Thermo Fisher Scientific, Waltham, MA, USA) in a microwave for 30 min to retrieve the antigens. Endogenous peroxidase activity was blocked with a 3% hydrogen peroxide (UltraVision Hydrogen Peroxide Block; Thermo Fisher Scientific) for 10 min. The sections were then incubated with a PTEN antibody (Santa Cruz, Santa Cruz, CA, USA) for 1 h at room temperature. PTEN immunoreactivity in sections was visualized using HRP polymer (UltraVision Quanto Detection System; Thermo Fisher Scientific) and DAB chromogen (DAB Peroxidase Substrate Kit; Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with Mayer’s hematoxylin (ScyTek Laboratories, Logan, UT, USA), dehydrated and then mounted using a mounting medium.

Standard protocols were used to prepare tissue microarray sections for immunohistochemistry. Then sections were blocked by UltraVision Hydrogen Peroxide Block (Thermo Scientific, CA, USA) and UltraVision Protein Block (Thermo Scientific), followed by primary antibodies (VEGF, 1:100) incubation. UltraVision Quanto Detection System horseradish peroxidase (HRP) Polymer (Thermo Scientific) and DAB Quanto (Thermo Scientific) were applied for staining, and hematoxylin was used for counterstaining. The score of erythema, scale and infiltration thickening of the skin lesions in mice was scored as 0 to 4: 0, negative; 1, weak; 2, moderate; 3, strong; and 4 very strong. Add the three scores to get the total points, taking the average of the scores of the mice in each group, and skin lesions in each group were observed.

Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue sections 3μm thin. Slides were placed for 20 minutes in 10mM sodium citrate buffer, pH 6.5 heat at 97°C in order to unmask antigens. To reduce nonspecific background staining due to endogenous peroxidase, specimens were incubated in UltraVision Hydrogen Peroxide Block (Thermo Scientific) for 10 minutes and after they were washed in Tris Buffer, for 5 minutes in Ultra Vision Protein Block (Thermo Scientific) to block nonspecific background staining. Sections were incubated for 60 minutes at room temperature with EF-1 alfa 2 (D-15 santa cruz-68481) to detect eEF1A2:sc-68481 mixed with EF-1 alfa1 (CBP-KK1): sc-21758 used at 1:300 dilution and after washed in Tris Buffer, for 30 minutes at room temperature with goat anti-rabbit IgG,F(ab’)2 –HRP: santa cruz-3837 used at 1:300 dilution. Finally, sections were incubated for 10 minutes with 3,3’ Diaminobenzidine chromogen (DAB Quanto–Thermo Scientific) and hematoxylin to nuclear counter stain.
As positive controls, we used staining intensity of eEF1A2 in normal breast parenchyma (external positive control) and staining intensity of eEF1A2 in normal breast parenchyma, if present in tumor tissue section (internal control).