Endogenous peroxidase 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. 

Immunohistochemistiy Tumor samples were fixed in formalin for 24 hours at RT and then embedded in paraffin. Serial paraffin sections of 3-4 pm were cut, placed on superffost plus slides, dewaxed with xylene, hydrated from ethanol to tap water, and transferred to PBS. Endogenous peroxidase activity was blocked by incubating slices with 3% H202 in PBS for 10 . 

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... 

Endogenous peroxidase activity not removed In this instance, staining will be observed in red and white blood cells. 

Inhibit endogenous peroxidase activity in the tissue sections by incubating in 0.3% H2O2 for 20 min... 

Place the sections in 0 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. 

Biopsy tissue specimens are fixed with formalin and embedded in paraffin. Sections (5 xm thick) are mounted on silanated slides, heated at 56°C for 1 hr, deparaffinized in xylene, rehydrated in graded ethanols, and rinsed in distilled water. They are placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for six cycles of 5 min each, followed by cooling at room temperature for 20 min. Endogenous peroxidase activity is blocked with hydrogen peroxide in distilled water for 8 min, and nonspecific background staining is prevented by treatment with nonimmune horse serum for 20 min. 

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. 

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). 

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. 

Tissue specimens are fixed with 4% formalin for 24hr and embedded in paraffin. Sections (4 xm thick) are mounted on poly-L-lysine-coated slides, deparaffmized in xylene, rehydrated in a descending series of ethanol, and dried for lhr at 60°C. Endogenous peroxidase activity is blocked with 1% H202 in methanol for 15 min. The slides are placed in a plastic jar filled with 0.01 M sodium citrate buffer (pH 6.0), which is placed in a microwave oven. They are treated at 750 W for three periods of 5 min each. During each heating cycle the buffer level is checked, and the evaporated portion is replaced with distilled water. 

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. 

J. J. Widmann, R. S. Cotran, and H. D. Fahimi, Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat fiver sinusoids by endogenous peroxidase activity, J. Cell Biol. 52 159-170 (1972). 

AP had not been used extensively in immunohistochemistry until publication of the unlabeled alkaline phosphatase-antialkaline phosphatase (APAAP) procedure (2, 3). The soluble immune complexes utilized in this procedure have molecular weights of approximately 560 kDa. The major advantage of the APAAP procedure compared to the earlier peroxidase techniques was the lack of interference posed by endogenous peroxidase activity. Because of the potential distraction of endogenous peroxidase activity, the alkaline phosphatase techniques were particularly recommended for use on blood and bone marrow smears. Endogenous alkaline phosphatase activity from bone, kidney, liver and some white cells can be inhibited by the addition of 1 mM levamisole to the substrate solution (4), although 5 mM has been found to be more effective (5). Intestinal alkaline phosphatases are not adequately inhibited by levamisole. 

Horseradish Peroxidase-Based Detection Methods Endogenous Peroxidase Activity... 

For practical purposes in immunohistochemistry, endogenous peroxidase activity can be defined as any activity that results in the decomposition of H202. Such activity is a common property of all hemoproteins such as hemoglobin (red cells), myoglobin (muscle cells), cytochrome (granulocytes, monocytes) and catalases (liver and kidney). Peroxidase activity may also be encountered in tissue areas adjacent to vascularized areas due to the diffusion of blood prior to fixation. 

Specimens rich in endogenous peroxidase activity may be processed using an alkaline phosphatase detection method instead of a peroxidase method, eliminating the background. 

Figure 1. Red blood cells showing endogenous peroxidase activity (a) before, and after blocking with three percent hydrogen peroxide, (b) Alkaline phosphatase-based detection methods.

Block endogenous peroxidase activity with methanol containing 0.5% H202 (100 volumes) for 30 min (see Note 6). Wash three times in distilled water and rinse in PBS, pH 7.4. 

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

Weir EE, Pretlow TG, Pitts A. Destruction of endogenous peroxidase activity in order to locate cellular antigens by peroxidase-labeled antibodies. J Histochem Cytochem. 1974 22 51. 

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