ABM’s Fast Red Violet Dye for Immunohistochemistry has been employed successfully as a rapid coupling diazonium salt for the detection of acid and alkaline phosphatases. The Fast Red Violet LB Salt solution is mixed with Naphthol AS Phosphate Solution and applied to tissue sections treated with an alkaline phosphatase labeled probe. Upon hydrolysis of the phosphate ester, the Fast Red Violet LB Salt solution reacts with the liberated Naphthol-AS forming a brilliant red-insoluble reaction product at the site of enzyme activity.
ABM’s Fast Red Violet Dye for Immunochemistry will contain 125ml of Naphthol-AS Phosphate solution (Reagent A) and 125ml of Fast Red Violet LB Salt solution (Reagent B).
After completing all immunologic incubations and washing steps, prepare the working reagent as follows while sections or membranes are still being rinsed: NOTE: Do not use phosphate containing buffers for rinsing. Inorganic phosphate inhibits alkaline phosphatases. 1. Determine the volume of reagent required for the procedure then remove the appropriate volume of Naphthol-AS Phosphate Solution, (Reagent A) and Fast Red Violet LB Salt Solution (Reagent B) and warm to room temperature. For example, if 10ml is needed, warm 5ml each of Naphthol AS Phosphate Solution and Fast Red Violet LB Salt Solution to room temperature. NOTE: Only warm up the amount of Reagent A and B to be used. Do not warm up all the reagents in the bottles as repetitive warming and cooling will decrease the stability of the reagents. 2. Immediately prior to use, mix equal volumes of Naphthol AS Phosphate Solution and Fast Red Violet LB Salt Solution together and mix thoroughly. 3. Completely cover tissue sections or membranes with the solution from step 2 and incubate at room temperature (22-27ºC) for 20 minutes. NOTE: The incubation time will vary according to the activity of the alkaline phosphatase and the temperature. The user must determine these parameters and standardize the method accordingly. 4. After 20 minutes (or some other predetermined incubation time) rinse sections or membranes thoroughly in deionized water to stop the reaction. Membranes may be dried after washing. 5. After washing sections thoroughly in deionized water, counterstain, if desired, in a progressive hematoxylin such as Mayer's or Gill No.2. Wash the sections following hematoxylin treatment in alkaline tap water. 6. The sections must be mounted in an AQUEOUS mounting medium. This is because Naphthol AS and Fast Red Violet LB reaction product are soluble in alcohols, xylene and xylene substitutes.
Suitable for the immunohistochemical localization of Alkaline Phosphatase labeled probes. Can be employed for localization of Alkaline Phosphatase labeled probes on Western, Northern, and Southern blots.
Store the Napthol-AS phosphate in refrigerator at 2-4ºC. Discard if solution becomes yellow or turbid. For the Fast Red Violet LB Salt solution, store at 2-4ºC. Discard if solution becomes turbid. Warm ONLY the amount of solution to be consumed to room temperature prior to use.
Depending on the activity of the alkaline phosphatase and the temperature, the incubation time of the sample with ABM’s Fast Red Violet Dye for Immunohistochemistry may vary and should be determined by the user.