1. Hydrolysis: The first step is acid hydrolysis, typically using hydrochloric acid (HCl). This process removes the purine bases from the DNA, creating apurinic acid without breaking the DNA's phosphodiester bond. The hydrolysis step is crucial as it creates aldehyde groups, which are necessary for the next step.
2. Schiff Reagent Application: Following hydrolysis, the sample is treated with Schiff reagent, which contains basic fuchsin that reacts with the aldehyde groups to produce a magenta color. This reaction is highly specific to DNA because RNA and other cellular components do not undergo the same hydrolysis process.
3. Washing and Counterstaining: The stained sample is then washed to remove any excess Schiff reagent. Counterstaining may be performed to provide contrast, making cellular structures easier to visualize.
