Periodic Acid-Schiff Staining of Cells: An In Vitro Technique to Detect Glycogen Levels in Peripheral Blood Mononuclear Cells
Transcripción
Peripheral blood mononuclear cells, or PBMCs, are circulatory blood cells with single, large nuclei, comprising B and T lymphocytes, natural killer cells, monocytes, and dendritic cells. PBMCs store surplus glucose in polymeric form as glycogen – a branched polysaccharide – in their cytoplasm.
To detect glycogen presence in PBMCs, prepare a uniform smear of isolated PBMCs on a glass slide. Treat the cells with formalin – a fixative solution, that cross-links cellular biomolecules, preserving PBMCs’ structural integrity.
Partially immerse the slide in amylase solution. This enzyme enters cells and hydrolyzes glycogen in the treated cells. This results in two distinct cell populations – enzyme-treated, glycogen-deficient, and non-amylase-treated, glycogen-rich cells.
Overlay them with periodic acid solution which oxidatively converts the hydroxyl groups of glycogen molecules to aldehydes.
Rinse the slide in water to halt the oxidation reaction and remove excess periodic acid. Treat the slide with Schiff reagent, which reacts with the aldehyde groups of periodic acid-treated glycogen, generating an insoluble magenta-colored complex.
Apply mounting media to restrain cells during imaging. Place a coverslip to spread the mounting media over the entire slide surface for better visualization. Image the stained cells.
Non-amylase-treated cells contain magenta glycogen granules, absent in enzyme-treated cells.
