Peroxidase, blocking

Deparaffmized, rehydrated, methanolic peroxidase-blocked (optional) tissue sections (see Chapter 12). 

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

Blood smears, tissue imprints, cell cultures and purified cells may be examined as fresh tissue or as fixed tissue. These cells can be centrifuged to make a pellet that is then fixed just as in tissue fixation. Alternatively, a fresh smear may be made on the slide, and the cells fixed either with acetone or 10% NBF for 10 minutes. It is important to incubate the slide with an endogenous peroxidase blocking solution... 

Block with three percent hydrogen peroxide or other peroxidase blocking reagent. Using a new bottle of hydrogen peroxide, perform a three percent H202 peroxidase block, followed by DAB and an appropriate counterstain. 

Peroxidase Block + Secondary Antibody + Streptavidin-HRP + DAB/AEC + Counterstain... 

Optimal fixation Peroxidase block Positive staining... 

Endogenous peroxidase activity not removed Perform peroxidase blocking step. 

In this bromoaspirin-inactivated structure, Ser , which lies along the wall of the tunnel, is bromoacetylated, and a molecule of salicylate is also bound in the tunnel. Deep in the tunnel, at the far end, lies Tyr, a catalytically important residue. Heme-dependent peroxidase activity is implicated in the formation of a proposed Tyr radical, which is required for cyclooxygenase activity. Aspirin and other NSAIDs block the synthesis of prostaglandins by filling and blocking the tunnel, preventing the migration of arachidonic acid to Tyr in the active site at the back of the tunnel. 

Propylthiouracil (PTU), but not methyl-mercaptoi-midazole (MMI), has an additional peripheral effect. It inhibits the monodeiodination of thyroxine to triiodothyronine by blocking the enzyme 5 mono-deiodinase [1]. In humans the potency of MMI is at least 10 times higher than that of PTU, whereas in rats PTU is more potent than MMI. The higher potency of MMI in humans is probably due to differences in uptake into the thyroid gland and subsequent metabolism, because in vitro inhibition of thyroid peroxidase by MMI is not significantly more potent than by PTU [1, 6]. Whether antithyroid drags have additional immunosuppressive actions is a matter of discussion [1, 2]. 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

The building blocks of proteins are the alpha-amino acids, and exclusively those with the L-configuration. There are 20 that occur in na- ture. They too all consist of the four elements C, H, N, and 0 two amino acids additionally contain sulfur (cysteine and methionine). In certain, but vital, enzymes (the peroxidases), sulfur is replaced by selenium. 

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. 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

D-tubocurarine from Chondrodendron tomentosum Ruiz et Pav. and Rauvolfia serpentina Benth. et Kurz and horse radish peroxidase 3 mg/ml, which prevent the peroxide formation, block the fruit formation as a whole, while yohimbine and gaillardine inhibit the seed formation. Neostigmine stimulates the fruit and seed yield, although its precursor physostigmine has no significant effect (Roshchina and Melnikova, 1998). 

The answer is c. (Katzung, pp 651-652.) Propylthiouracil is a thioamide that interferes with the production of thyroid hormone. Its primary action is prevention of thyroid hormone synthesis by blocking thyroid peroxidase catalysis leading to interference with iodine organification. 

PTU and MMI block thyroid hormone synthesis by inhibiting the peroxidase enzyme system of the thyroid gland, thus preventing oxidation of trapped iodide and subsequent incorporation into iodotyrosines and ultimately iodothyronine ( organification ) and by inhibiting coupling of MIT and DIT to form T4 and T3. PTU (but not MMI) also inhibits the peripheral conversion of T4 to T3. 

When using high-temperature antigen retrieval, you may skip blocking alkaline phosphatase, since some endogenous enzymes, such as alkaline and acid phosphatases, in contrast to peroxidase, are destroyed by boiling for even a short time at 100°C (Cattoretti et al. 1993). 

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 . 

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