Direct Blue 1 (Pontamine sky blue), a counterstain for background autofluorescence in immunofluorescence histochemistry, structurally resembles glutamate and operates as a potent, competitive VGLUT inhibitor, not impacting plasma membrane transporters. This compound stands out as the inaugural small molecule PrP ligand to inhibit Aβ binding.

Instructions
I. Dissolve and prepare dye solution
1. Solution preparation: Dissolve Direct Blue 1 in water. Common dye concentrations range from 0.1% to 1% (w/v). The specific concentration is adjusted according to experimental needs. For example, when the dye solution concentration is 0.5%, 5 mg of dye is usually mixed with 1 mL of solvent.
2. Staining process:
1) Prepare samples: Prepare samples to be stained (such as textiles, proteins, cells, etc.) according to the type of experiment.
2) Staining solution: Add the prepared Direct Blue 1 solution to the sample. The staining time is usually 20 to 60 minutes, depending on the staining intensity requirements.
3) pH value: During staining, the pH value of the dye solution usually needs to be kept at acidic or neutral (pH 4-7). If the sample is sensitive to pH, precise pH control may be required.
4) Washing: After staining, wash with water or an appropriate buffer to remove unbound dye until the sample no longer releases dye.
II. Applications
1. Protein or DNA staining: Direct Blue 1 can be used for staining in gel electrophoresis, especially for staining proteins and other biomacromolecules.
2. Cell staining: In cell biology experiments, Direct Blue 1 is often used for cell staining to facilitate observation of cell morphology or counting under a microscope.
3. Surface adsorption experiments: Because Direct Blue 1 can strongly adsorb to different surfaces (such as fibers, membranes or cell surfaces), it can also be used for surface adsorption or adsorption experiments.