Periodic Acid-Schiff Staining of Cells: An In Vitro Technique to Detect Glycogen Levels in Peripheral Blood Mononuclear Cells

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.

In this step, place the slide on a flat surface. Apply 2 milliliters of Periodic Acid solution on the sample, and incubate for 5 minutes at room temperature. Then, rinse the slide several times with distilled water. Apply 2 milliliters of Schiff's reagent on the slide, and incubate at room temperature for 15 minutes.

Subsequently, wash the slide with distilled water for 5 minutes, and then, leave it to air-dry. Next, apply 100 microliters mounting media on the slide, and cover it with one large coverslip, or apply 50 microliters and use two small coverslips. After that, apply clear nail polish on the edges of the coverslip. Let them dry overnight.

Then, obtain images with the Binocular Light microscope using the 100X objective.