During this protocol, scientists prepare a solution with BCIP, NBT, levamisole and the enzyme alkaline phosphatase buffer at pH 9.5. AP buffer contains tris(hydroxymethyl)aminomethane, magnesium chloride, the polysorbate detergent Tween 20 and sodium chloride. The solution is incubated at room temperature for 24 hours and is then available to use.
Detection of mRNA with alkaline phosphatase is a protocol used to study the nucleic acids mRNA and DNA in animal embryos. During this procedure, BCIP/NBT is used as a substrate for the enzyme alkaline phosphatase. BCIP/NBT is added to the embryos after they are rinsed twice in alkaline phosphatase staining buffer. Nucleic acid sites appear in purple.
During this protocol, technicians separate and identify the components of proteins using electrophoresis gel apparatus and a technique called Western blotting, which requires the use of a piece of equipment containing a nitrocellulose membrane and specific antibodies. After standard electrophoresis, technicians perform the Western blotting protocol, incubating the membranes for 30 minutes in a buffer solution and at room temperature. The antibody p47-phox is added and incubated for one hour. Conjugated alkaline phosphatase antibody is added, and after one hour, also BCIP/NBT. The reaction is stopped after 15 minutes of incubation by rinsing the membrane with water.
Medaka is a small fish that produces eggs daily and is widely used in embryonic stem cell research. According to Kursad Turksen in "Embryonic Stem Cell Protocols: Isolation and Characterization," Medaka eggs are incubated in embryo medium at 78.8 degrees Fahrenheit for seven days. Embryos are then washed in phosphate buffered saline solution, fixed in a methanol-acetone mix and stained with BCIP/NBT. Samples are then kept in the dark for 24 hours at 82.4 degrees Fahrenheit. The cells chromosomes are then observed under a microscope.