Peroxidase quenching endogenous activity

When using HRP as an enzyme marker, incubate the sections for 15 min in 0.3% H2O2 in either methanol or water to quench endogenous peroxidase (see Sect. 5.2). If endogenous peroxidase activity does not present a problem, this step may be omitted. 

Overdigestion of tissue is common when proteinase-K or triton is used to improve antigen retrieval penetrance of the primary antibody. The easiest correction is to dilute triton solutions and decrease the time of the proteinase-K incubation. Tissue can also be digested when the hydrogen peroxide solution, used to quench endogenous peroxidase activity, is left on too long or is too concentrated. To correct this, check... 

Tissue specimens are fixed with 4% paraformaldehyde and embedded in paraffin at 60°C for 1 hr. Sections (4 pm thick) are mounted on gelatin-coated glass slides, deparaffinized, and rehydrated in distilled water. They are treated with 0.005% pepsin for 15 min at 37°C, followed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave oven (300 W) at 80°C for 15 min. The sections are washed in distilled water for 5 min, rinsed in 0.01 M PBS (pH 7.2) for 15 min, and treated with 0.3-1% H202 to quench endogenous peroxidase activity. 

The culture is incubated in methanol containing 3% H202 for 10 min to quench endogenous peroxidase activity. The cells are rehydrated in 70% ethanol for 2 min, followed by rinsing for 5 min in PBS. Nonspecific binding is blocked by incubating the cells for 1 hr in the blocking buffer (1% BSA in PBS). 

Fresh rat brain tissue is immediately immersed in 2-methyl butane at -15 to -25°C for several minutes and stored at -70°C. Sections (10 p,m) are cut in the parasagittal plane on a cryostat, which are thaw-mounted onto poly-L-lysine-coated glass slides and stored at -70°C. The sections are dried on a slide warmer at the lowest setting to avoid excessive drying. They are fixed with 4% formaldehyde in PBS (pH 7.5) for 30 min and then rinsed three times for 10 min each in PBS to remove excess fixative. This is followed by treatment with 0.3% hydrogen peroxide in PBS for 30-40 min to quench endogenous peroxidase activity. 

Incubate sections in 2% hydrogen peroxide in methanol for 5 min to quench endogenous peroxidase activity. 

Endogenous peroxidase activity is quenched by treatment with 0.5% H202 in methanol, immunostaining is carried out using a Vectastain Elite ABC kit, and visualization of the secondary antibody is achieved with DAB enhanced with nickel (Vector Laboratories). 

Tissues are fixed with 10% neutral phosphate-buffered formalin and embedded in paraffin, and sections (5 pm thick) are mounted on poly-L-lysine-coated slides heated at 60°C for 30 min. The sections are deparaffinized in four changes of xylene and then rehydrated in a descending series of ethanol. Endogenous peroxidase activity is quenched by immersing the sections in 1% H202 in distilled water for 5 min. The sections are rinsed in three changes of distilled water, transferred to a moist chamber, and covered with PBS. 

Unquenched endogenous alkaline phosphatase activity may be seen in leucocytes, kidney, liver, bone, ovary bladder, salivary glands, placenta and gastro-intestinal tissue. Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme label such as horseradish peroxidase. Intestinal alkaline phosphatase is not quenched by the addition of levamisole. Pretreat the tissue with 0.03 N HCI. 115-121... 

Endogenous peroxidase activity may also be quenched by immersing sections in 0.1% phenylhydrazine m PBS for 5 min at room temperature This is reportedly the most gentle treatment (37), and is thus recommended for labile antigens and cryostat sections. 

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