MH/MHS/RnD Cerebellum

Hematoxylin & Eosin

The cerebellar cortex is composed of the outer, hypocellular molecular layer and an inner, densely populated granular cell layer. At the interface of these two layers are large neurons known as Purkinje cells.

At higher magnification, examine Purkinje cells stained with hematoxylin and eosin (H&E).

• Their nuclei are much larger compared to those in the inner, densely populated granular layer.
• Their cell bodies and dendrites are intensely acidophilic. This indicates they contain large amounts of proteins.

Only the nuclei of neurons in the inner, densely populated granular layer are visible.

Toluidine Blue

Toluidine blue is a basic dye that binds nucleic acids but preferentially stains RNA.

At higher magnification in the cerebellum, examine Purkinje cells stained with toluidine blue.

• Their large nuclei contain lightly stained euchromatin.
• Their nucleoli are intensely stained because they contain negatively charged RNA involved in ribosome assembly.
• Nissl (i.e., chromophil) substance appears dark blue due to the staining of ribosomal RNA, giving the cytoplasm a mottled appearance.

The prominent nucleoli and Nissl substance indicates these cells synthesize large amounts of protein.

Golgi's Method

Golgi's method randomly stains a limited number of neurons and their processes (axon and dendrites). This allows the morphology of individual neurons to be investigated.

At higher magnification in the cerebellum, examine the morphology of Purkinje cells (#1, #2, #3):

• Perikaryon (cell body) - large diameter
• Dendrites - highly branched in the outer molecular layer of the cerebellum
• Axon - a single axon extends from each Purkinje cell into the inner granular layer

Immunohistochemistry

Purkinje cells can also be identified by using immunohistochemistry for proteins expressed in these cells. Heat shock protein H1 (HSPH1) is involved in the folding of proteins and degradation of misfolded proteins. It is highly expressed in Purkinje cells.

HSPH1 was detected by incubation with the following antibodies and dyes:

• Anti-HSPH1 goat primary antibody that binds to HSPH1 in Purkinje cells
• Horseradish peroxidase (HRP)-conjugated anti-goat secondary antibody that binds to the bound goat primary antibody
• Diaminobenzidine (DAB) that is oxidized by HRP into a brown precipitate
• Hematoxylin (blue/purple) to counter stain nuclei and other cells

The brown precipitate is found in the cell bodies and dendrites of Purkinje cells (#1, #2, #3). The light brown staining of the outer molecular layer of the cerebellum is non-specific.

Courtesy of Alexander E. Kalyuzhny (R&D Systems, Minneapolis, MN).

Immunofluorescence

Purkinje cells can also be identified by using immunofluorescence for specific proteins. The bound primary antibody is visualized with a secondary antibody conjugated with a fluorescent dye. Multiple proteins can be labeled in the same specimen using multiple primary antibodies and secondary antibodies conjugated with different dyes. Upon illumination, the fluorescent dyes will emit different colors of light.

• Purkinje cells (green)
• Glia (non-neuronal cells; red)
• Nuclei (blue)

Courtesy of Thomas Deerinck (National Center for Microscopy and Imaging Research, University of California, San Diego, CA).

Lucifer Yellow Injection

The morphology of cells can also be examined by using a small electrode to inject a fluorescent dye into individual cells.

A Purkinje cell in the cerebellum was injected with the fluorescent dye Lucifer Yellow. This image was generated from a series of optical sections acquired with a laser scanning confocal microscope.

• Perikaryon (cell body) - large diameter
• Dendrites - highly branched in the outer layer of the cerebellum
• Axon - a single axon extends into the inner layer of the cerebellum

Courtesy of Maryann Martone and Eric Bushong (University of California, San Diego, CA).