Endogenous peroxidase

Block sections for 10 15 min before or after primary antibody incubation. 

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Jylling AMB, Lindebjerg J, Nielsen L, et al. Immunohistochemistry on frozen section of sentinel lymph nodes in breast cancer with improved morphology and blocking of endogenous peroxidase. Appl. Immunohistochem. Mol. Morphol. 2008 16 482-484. 

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. 

Incubate sections with 3% H2O2 in PBS for 10 min to block endogenous peroxidase. 

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... 

Again, check the dilutions of the primary and secondary antibodies and reevaluate tbe incubation times of the primary, secondary, tertiary complex, and chromagen steps. Change blocking sera used to reduce nonspecific background and/or include blocking serum in primary and secondary antibody solutions. Ensure that the endogenous peroxidase is sufficiently quenched. 

Block endogenous peroxidase and peroxidase-like activity by incubation in 1.5% H2O2 in PBS endogenous enzyme blocking solution for 15 min with constant stirring (see Note 8). 

The enzyme or the chromogen detection system determines whether any endogenous material must first be destroyed. If a peroxidase marker molecule is to be used, endogenous peroxidase or peroxidase-like activity should be blocked. Because these preparations are more fragile than a fixed embedded sample, endogenous enzyme is inactivated with a weaker blocking solution than would... 

Optional endogenous peroxidase blocking with methanolic peroxide can be performed either before or after HIER. 

Block endogenous peroxidase in methanol containing 0.5% hydrogen peroxide for 15 min. 

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. 

Sodium azide/hydrogen peroxide solution blocks endogenous peroxidase enzyme activity This is important m cervical smears where large numbers of neutrophil polymorphs and red blood cells are present. Although often not essential when analyzing cultured cells or biopsies, incubation m this solution is equally effective with these sample types. 

As the TSA-ABC protocol dramatically improves the signal intensity by the peroxidase-catalyzed deposition of biotinylated tyramide, blocking of endogenous peroxidase is required. To ensure quenching of the residual peroxidase, the use of a higher concentration and duration of treatment with H202 is recommended. 

In contrast to the EPOS system, a modification of the highly sensitive two-step immunohistochemical EnVision system allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min (Kammerer et al., 2001). In this study 38 out of 45 antibodies tested showed specific staining. In fact, the modified EnVision procedure allows the use of any suitable primary antibody, preferably monoclonal antibodies. Like the EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidase molecules for detection. No attempt was made to block endogenous peroxidase, nor was any antigen retrieval pretreatment used. Because of the very short incubation durations, a humid chamber is not required to avoid evaporation of immunoreagents. 

When the DAB method is used, inhibit endogenous peroxidase by treating the sections with 500 xl of 30% H202 in 50 ml of PBS for 30 min, followed by thorough washing in PBS. 

Surgically removed tissues are fixed with 4% buffered formalin and embedded in paraffin (Turner et al, 1999). Sections (4 p.m thick) on slides are deparaffinized, rehydrated, and then treated with 3% H202 to block endogenous peroxidase. They are placed in 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven. An EPOS rabbit antihuman Ki-67 antibody is applied as supplied (DAKO) it is ready to be used without any dilution. Color development is accomplished with metal-enhanced DAB for 15 min, followed by light counterstaining with hematoxylin. Quantification of Ki-67 antibody-labeled... 

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. 

The sections are treated with Biotek enzyme (Ventana Biotek) for 10 min, followed by blocking of endogenous peroxidase with 3% hydrogen peroxide in buffer for -8 min. Immunostaining is carried out in an automated immunostainer (TechMate 1000 Equipment Co., Shelton, CT). The slide rack is sonicated for 1 min and then cooled in buffer in the staining container for an additional 10 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. 

Sections are covered with cover-slips, sealed with rubber cement, denatured by heating at 78°C in a water bath for 10 min, and hybridized overnight at 37°C. The coverslips are carefully removed by floating the slides in 2 X SSC, and the sections are washed twice for 10 min each in a mixture of 2 X SSC and 50% formamide at 40°C, and then three times in PBS. Endogenous peroxidase is blocked by incubation in 1% H202 for 15 min. 

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. 

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