Frozen section NADH myocardial flavin activity staining kit product manual (Chinese version)
The main purpose
The frozen section NADH myocardial flavin activity staining reagent is a kind of intention to use the artificial electron acceptor dye dimethylthiazole diphenyltetrazole olfactory blue (MTT), which is catalytically reduced by NADH diaphorase to produce an insoluble blue-black pigment precipitate. An authoritative and classic technical method for analyzing enzyme activity in frozen tissue samples. The technology has been proven through careful improvement of traditional methods and successful experiments. It is mainly used for the qualitative detection of diaphorase activity in animal tissue cells. The product is strictly sterile, ready to use, simple in operation, stable in performance and clear in color.
technical background
Diaphorase, also known as lipoamide dehydrogenase, diaphorase, dihydrolipoamide dehydrogenase, dihydrolipoyl dehydrogenase, etc. . Because it is extracted from pig myocardium early, the product shows fluorescent yellow-green protein, so it is called diaphorase. The molecular weight is 102,000 to 110,000. NADH diaphorase directly reacts with Flavian protein to release hydrogen atoms and reduces oxidized flavin adenine dinucleotide (FAD) to reduced flavin adenine dinucleotide (FADH2). Hydrogen atoms give oxidized nicotinamide adenine dinucleotide (NAD) to form reduced nicotinamide adenine dinucleotide (NADH), which acts as an electron donor to reduce artificial electron acceptors, such as the dye tetrazolium (tetrazolium) compounds, including Nitro Blue Tetrazolium (NBT) and dimethylthiazole diphenyltetrazole olfactory blue (3-[4,5-dimethylthiazol-2-yl]-2,5- Diphenyl-tetrazolium bromide; MTT), which produces insoluble nail pigment and is used to identify various animal tissues such as visceral nerve tissue.
product content
Cleaning solution (Reagent A) 100 ml
Staining solution (Reagent B) 10 ml
Reaction solution (Reagent C) 1 ml
Fixative (Reagent D) 10 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; transfer 900 μl of the stain solution (Reagent B) to a new 1.5 ml centrifuge tube, add 100 μl of the reaction solution (Reagent C) , and mix. After that, mark it as a dyeing working solution and place it in an ice bath to avoid light. Then do the following.
- Take out the 10 micron thick frozen tissue section to be tested
- Carefully add 200 μl of cleaning solution ( Reagent A ) over the slice to cover the entire sample surface.
- Carefully remove the cleaning solution from the slice ( Reagent A )
- Carefully add 200 μl of staining solution to cover the entire surface of the sample
- Incubate in a 37 ° C incubator for 30 minutes to avoid light
- Carefully remove the staining solution from the slice
- Carefully add 200 μl of cleaning solution ( Reagent A ) to the slice and spread the entire surface of the sample.
- Carefully remove the cleaning solution from the slice ( Reagent A )
- Repeat the experiment steps 6 to 8 times
- Carefully add 200 μl of Fixative ( Reagent D ) on the slice to cover the entire surface of the sample.
- Incubate for 15 minutes at room temperature
- Carefully remove the fixative from the slice ( Reagent D )
- Carefully add 200 μl of cleaning solution ( Reagent A ) to the slice and spread the entire surface of the sample.
- Carefully remove the cleaning solution from the slice ( Reagent A )
- Repeat the experimental steps 12 to 14 times
- Put a cover slip or cover (neutral resin)
- Immediately under light microscope observation and counting: tissue cells expressing NADH diaphorase are positive tissue cells, showing blue-black
Precautions
- This product is 50 operations
- Wear gloves when handling
- Reagent C avoids repeated freezing and thawing
- In the negative control, the dyeing working solution does not contain the reaction solution (Reagent C)
- Myocardial tissue can be used for positive control
- 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 5 to 60 minutes
- Immediately after the dyeing, optical microscopy
- Save the sample after dyeing to avoid illumination
- The dyed reference image is shown below
The company provides NADH myocardial yellow enzyme activity NBT staining reagent products
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
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