Whole tissue cytochrome oxidase activity staining kit instructions

Whole tissue cytochrome oxidase activity staining kit product manual (Chinese version)

The main purpose

Whole tissue cytochrome oxidase activity staining reagent is a non-enzymatic natural oxidation of cytochrome C catalyzed artificial electron donor dye diaminobenzidine to form a brown insoluble product to analyze cytochromes in whole tissue samples. An authoritative and classic technical approach to oxidase activity. This technology has been proven through careful improvement of traditional NADI methods and successful experiments. It is mainly suitable for the qualitative detection of cytochrome oxidase activity in various animal tissues. The product is strictly sterile, ready to use, simple in operation, stable in performance and clear in color.

technical background

Cytochrome oxidase is a general term for the enzyme portion of the oxidative respiratory chain. It is present on the mitochondria of eukaryotic cells and supplies energy to cells primarily through oxidative phosphorylation. Diaminobenzidine (DAB) is an artificial electron donor dye that produces non-enzymatic natural oxidation while providing electrons by physical binding to metalloproteins such as ferritin-containing cytochrome C. (Cytochrome C acts as an electron acceptor) in the electron transport system of the respiratory chain, thereby exhibiting color changes and deposition, forming a tan insoluble product, and is not affected by ethanol. At the same time as the DAB is naturally oxidized, active oxygen, such as hydrogen peroxide, is generated, which is prevented from accumulating by catalase. In the presence of a large amount of oxidized DAB, ferrous cytochrome C is oxidized to normal iron cytochrome C by the action of cytochrome oxidase. The oxidized DAB tan insoluble product was thus used to detect the activity of cytochrome oxidase in tissue cells.

product content

Cleaning solution (Reagent A) 200 ml

Dyeing Liquid A (Reagent B1) 40 ml

Dye B (Reagent B2) 4 bottles

Reaction solution (Reagent C) 3 ml

Fixative (Reagent D) 30 ml

Product manual 1 copy

storage method

Store the staining solution (Reagent B) and the reaction solution (Reagent C) in a -20 °C refrigerator to avoid light; the rest are stored in a 4 °C refrigerator to ensure June.

User-supplied

1.5 ml centrifuge tube: container for working fluid preparation

Incubator: for reaction incubation

Neutral resin: used for slicing

Optical microscope: used for observation and analysis after sectioning

Experimental procedure

Before the start of dyeing, melt the reagent in the -20 °C refrigerator at room temperature; remove 9 ml of staining solution A (Reagent B1) into 1 bottle of staining solution B (Reagent B2) , mix and mark as dyeing working solution . Place in the ice tank for use, avoiding light (note: valid for 1 week); continue to transfer 2.7 ml of dyeing working solution to a new 1.5 ml centrifuge tube, add 300 μl of reaction solution (Reagent C) , mix and mark, mark The reaction working solution is placed in an ice bath to avoid light. Then do the following.

• Prepare a 6-well cell culture plate
• Place freshly excised animal tissue samples (Note: The recommended tissue size is 0.5 cm thick and 1 cm long; if it is too large, it is best to cut it )
• Flatten the tissue
• Carefully add 3 ml of cleaning solution ( Reagent A ) to clean tissue samples
• Carefully remove the cleaning solution
• Carefully add 3 ml of reaction working solution to submerge the entire tissue
• Put in a 37 ° C incubator and incubate for 60 minutes to avoid light
• Carefully remove the reaction solution
• Carefully add 3 ml of cleaning solution ( Reagent A ) to clean tissue samples
• Carefully remove the cleaning solution
• Repeat the experiment steps 9 to 10 times
• Carefully add 3 ml of fixative ( Reagent D ) to submerge the entire tissue
• Incubate for 15 minutes at room temperature
• Carefully remove the fixative
• Carefully add 3 ml of cleaning solution ( Reagent A ) to clean tissue samples
• Incubate for 15 minutes at room temperature
• Carefully remove the cleaning solution
• Repeat the experimental steps 15 to 17 times
• Subsequent paraffin sections (6 microns thick) or frozen sections (10 microns thick)
• Observation and counting under a light microscope: Tissue cells expressing cytochrome oxidase activity were positive tissue cells and appeared brown.

Precautions

• This product is 10 operations
• Wear gloves when handling
• Reagent B and Reagent C avoid repeated freezing and thawing
• Dyeing working solution should not be stored for a long time, preferably used within 1 week.
• In the negative control, the GENMEN reaction working solution may contain sodium azide.
• Keep the sliced ​​surface basically dry each time the reagent solution is changed
• When the reagent solution is slicing the surface, avoid the presence of air bubbles while ensuring that the surface of the slice is covered.
• The entire operation, in the dark state
• Staining incubation time, adjusted according to sample and enzyme activity, usually 1 to 3 hours
• Immediately after the dyeing, optical microscopy
• Save the sample after dyeing to avoid illumination
• Cytochrome oxidase activity staining reference image:

The company provides a series of tissue cell enzyme activity dyeing reagent products

Quality Standard

• This product has been certified to be stable.
• This product has been identified and clearly colored

Pharmaceutical Packaging

pharmaceutical blister Packaging