Factors that affect staining include:
Concentration of the Dye - The greater the concentration of the dye, the more the dye is bound to tissue components.
Temperature - An increase in temperature increases the rate at which the dye diffuses throughout the tissue sample. Temperature can also alter tissue components so that they are more receptive to dye penetration.
pH of the Staining Solution - Cells and other tissue elements often have an affinity for stains/dyes with specific pH ranges. Thus, the pH of the staining solution can have a direct impact on the ability of a dye to bind with its intended tissue element.
Tissue fixation - Fixation alters and reorganizes certain molecular structures in tissue samples so that they have an increased permeability and are more receptive to staining. Unfixed tissue elements have limited binding sites for dyes.