Pathological experiment TUNEL detection
TUNELASE (TDT) -Mediated Dutp Nick End Labeling, end deoxycodide transferase mediated DUTP gap end marker assay), also known as the in situ tag method of DNA break. The breakage of chromosome DNA in apoptosis is a progressive stage of step, and the chromosome DNA is first decreased to 50-300 kB of a large fragment of the endogenous nucleic acid hydrolase, and then about 30% of the chromosome DNA in Ca2 + and Mg2 + dependent nucleic acid endonuclease, is randomly cut between the nucleometric unit, forming 180 to 200 BP nucleom DNA polymer. DNA double strand break or a series of DNAs generated by a gap in a chain, a series of DNAs 3 u0026 RSQUO; -OH ends can be deoxyribonucleotides and fluorescene under the action of deoxyribonucleotide terminal transferase (TDT). Peroxidase, alkaline phosphatase or biotin formation of derivatives labeled DNA 3 u0026 RSQUO; - end, re-use horseradish peroxidase labeled streptavidin and derivative biotin (each A brown hydrogen peroxide is oxidized, and the cyclization reaction is oxidized, the hydrogen peroxide, and the hydrogen peroxide, the hydrogen peroxide, and the hydrogenation reaction, forming a phenyl hydrazine polymer to show tan, final The apoptosis occurred by counting the ratio of TUNEL positive cells in different field of view on each slice. Since normal or proliferative cells have almost no DNA break, there is no 3 u0026 rsquo; -oh formed, which is rarely dyed.
(1) after collecting processing Cell or paraffin slice deprnowled to water. (2) dip in hydrogen peroxide, inhibiting endogenous catalase.
(3) incubated with protease K for 15 to 30 minutes.
(4) Tuxel reaction mixture (i.e., use, 4 O
C-prototype), incubated for 60 minutes, under fluorescent mirror (green) analysis results. (5) Add transformant -POD, incubate for 20 to 30 min.
(6) Add a DAB substrate solution, incubate for 5 to 10 min. (7) Neutral gum or glycerol seal, and the results of the light microscope. Service Description (1) Customer provides: Cultivated cells and drugs required; or provide paraffin specimens
(2) Company provides: BasicExperimental steps, pictures, data analysis results, surplus drugs
(3) Experimental cycle: 1 weekSpecific needs to be determined according to cell growth and experimental content.