MH 003 Toluidine Blue

Cerebellum

Toluidine blue is a basic dye that binds nucleic acids, but preferentially stains RNA. When it stains structures blue, it is called orthochromatic.

At the interface between the two layers of the cerebellum, a layer of large cells known as Purkinje cells can be found. At high magnification, examine individual Purkinje cells sectioned through their nuclei and dendrites.

• The nucleolus is intensely stained (i.e., chromophilic) because it contains negatively charged RNA involved in ribosome assembly.
• Nissl (or chromophil) substance in the cytoplasm contains negatively charged RNA found in free ribosomes and ribosomes bound to endoplasmic reticulum.
• The presence of chromophil substance suggests these cells synthesize large amounts of protein.

Liver

The liver is composed primarily of hepatocytes. Examine the staining of individual hepatocytes at high magnification.

• The cytoplasm of hepatocytes contains the most intense staining (i.e., chromophilic) around the periphery of the nucleus.
• Most hepatocytes contain a single nucleolus, but careful examination shows many cells have multiple nuclei.

Pancreas

Most of the pancreas is composed of acinar cells. Acinar cells are polarized cells clustered around a central lumen which is the entrance to a duct (i.e., an acinus).

• The apical cytoplasm near the lumen contains unstained secretion granules. They are released from the apical surface into the central lumen.
• The basal portion of the cell contains the nucleus with a prominent nucleolus, cytoplasm with abundant chromophil substance, and is free of secretion granules.
• The intense staining in the cytoplasm contains negatively charged RNA found in free ribosomes and ribosomes bound to endoplasmic reticulum.
• The presence of chromophil substance suggests these cells synthesize large amounts of protein.

Lymph Node

Lymph nodes contain many lymphocytes. Note the scattered larger cells with more chromophilic cytoplasm, these are macrophages.

• Lymphocyte nuclei are almost completely heterochromatin with small nucleoli.
• The light staining of the cytoplasm in lymphocytes suggests these cells are not actively synthesizing much protein.

Mast Cells

Toluidine blue is often used to identify mast cells by virtue of heparin (which is very polyanionic) found in their secretion granules. In the connective tissue surrounding the lymph node, several mast cells can be found.

• The secretion granules of the mast cells are stained a dark purple.
• This is an example of metachromisia which occurs when the color of a dye shifts with staining (in this case, from blue to dark purple).