Peroxidase is an enzyme abundantly present in liver, kidney, neutrophils and epithelial cells of small intestinal mucosa, more present in the peroxisomes of cells, participating in various oxidative reactions in cells, which can oxidize various substrates.

Principle

The basic principle of the experiment is that peroxidase is a rich enzyme in liver, kidney, neutrophils and small intestine mucosal epithelial cells, more in the peroxisomes of the cell, involved in various oxidation reactions in the cell, can oxidize various substrates. This experiment uses the above properties of peroxidase, the substrate _{H2O2 decomposition} , the production of new oxygen, so that the colorless benzidine oxidation into blue benzidine blue, and then into a brown product, can be based on the color reaction to determine the presence or absence of peroxidase or how much.

Operation method

Experiments showing peroxidase in cells

Principle

The basic principle of the experiment of peroxidase display in cells is that peroxidase is an abundant enzyme in liver, kidney, neutrophil and small intestine mucosal epithelial cells, more exist in the peroxisome of the cell, participate in various oxidation reactions in the cell, and can oxidize various substrates. This experiment uses the above properties of peroxidase to decompose the substrate H2O2, generate new oxygen, oxidize the colorless benzidine into blue benzidine blue, and then into brown products, which can be based on the color reaction to determine the presence or absence or amount of peroxidase.

Materials and Instruments

Equipment:
Staining cylinder, slides, coverslips, pipettes, syringes, absorbent filter paper, dissecting equipment, wax discs, ordinary light microscope, mice.
Reagents:
① Benzidine mixture (benzidine (4,4-diamino benzidine) 0.2 g, 95% ethanol 100 mL, 3% hydrogen peroxide, 2 drops . (This solution is prepared for use); ② 0.5% sulfate solution (0.5%)
② 0.5% copper sulfate; ③ 1% copper sulfate
③1% red
④Neutral gum

Move

The basic procedure of the peroxidase display experiment in cells can be divided into the following steps:
A. Take 1 mouse , execute it by cervical dislocation method, quickly dissect the hind limb to expose the femur, cut the femur from one end, and suck out the bone marrow drop to one end of the slide with a syringe (1 drop of PBS can be added to dilute it if necessary).
B. Push the slides, and air dry them at room temperature.
C. Immerse the smears into 0.5% copper sulfate for 30 s .
D. Immerse in benzidine mixture for 6 min.
E. Rinse under running water and re-stain in 1% saffron solution for 2 min.
F. Rinse under running water and dry at room temperature.
G. Microscopic examination or 1 drop of neutral gum, and then cover the film with a cover slip for sealing and observation.

Caveat

1. The benzidine mixture in this experiment is very easy to be oxidized in the air and show brown color, which reduces the dyeing effect, therefore, the solution is ready to be prepared when applying, and attention should also be paid to reduce the contact with the air in the operation process.

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