Secondary anti mouse antibody hrp

Paraffin embedded tissue slides were deparaffinised and rehydrated, endogenous peroxidise activity was blocked with 3% Hydrogen peroxide, antigen retrieval was performed in 10 mmol/L citrate buffer, and nonspecific binding was blocked with blocking reagent. Anti-PXDN antibody (NBP1-84316, Novus Biologicals, USA), anti-NTN4 antibody (NBP2-13680, Novus Biologicals, USA) and anti-GLIS3 antibody (NBP2-16668, Novus Biologicals, USA) was applied at 5 μg/mL, 1 μg/mL and 8 μg/mL concentrations, respectively and incubated overnight at 4°C, followed by 60 minute incubation with secondary anti-mouse antibody HRP (Dako). The chromogen used was 3-amino-9-ethylcarbazole (AEC). Human placenta was used as the positive control for PXDN and NTN4. Human prostate tissue was used as positive control for GLIS3. Negative control for which the primary antibody was substituted with the same concentration of mouse IgG was also used for all antibodies tested. Slides were scanned using a ScanScope XT (Aperio) and immunohistochemical reactivity was evaluated by two independent investigators.

Paraffin embedded tissue slides were deparaffinised and rehydrated, endogenous peroxidise activity was blocked with 3% Hydrogen peroxide, antigen retrieval was performed in 10mmol/L citrate buffer, and nonspecific binding was blocked with blocking reagent. THBS1 antibody (A6.1, NB100-2059, Novus Biologicals, USA) was applied at 6μg/mL concentration and incubated overnight at 4°C, followed by 60 minute incubation with secondary anti-mouse antibody HRP (Dako). The chromogen used was 3-amino-9-ethylcarbazole (AEC). Human placenta was used as the positive control for THBS1 and a negative control, for which the primary antibody was substituted with the same concentration of mouse IgG. Slides were scanned using a ScanScope XT (Aperio) and immunohistochemical reactivity was evaluated by two independent investigators. The expression of THBS1 was categorized into four grades. They were arbitrarily scored as 3, strong staining; 2, moderate staining; 1, weak staining and 0, no staining.