The neuronal cell body contains a large nucleus with a folded nuclear membrane, sparse chromatin, and a distinct nucleolus. Within the cell body, the cytoplasm is filled with Nissl granules—basophilic granules that represent the rough endoplasmic reticulum and produce a characteristic spotted basophilic appearance in many neurons. Nissl granules can be visualized using various stains, such as neutral red, methylene blue, toluidine blue, and methyl violet. Variations in staining protocols, pH values, and differentiation times allow some stains to either specifically highlight Nissl substance or simultaneously visualize neuronal nuclei and neuroglia. Nissl bodies, also known as Nissl substance, are triangular or oval granular structures distributed in the cytoplasm of neurons. They can be stained purple-blue by basic dyes including thionine, methylene blue, toluidine blue, and cresyl violet. All types of neurons contain Nissl bodies, but their shape, quantity, and distribution often vary. Nissl bodies are also present in dendrites but are absent from axons and the axon hillock of the cell body. Nissl bodies undergo changes in response to the physiological state of neurons: as important sites for protein synthesis in neurons, their volume decreases significantly when neurons are stimulated.
Nissl Staining Solution (Cresyl Violet Method) uses cresyl violet as the core dye. Cresyl violet is light-sensitive and can well demonstrate the changes of Nissl bodies. It has the main advantages of simple operation, stable staining, and broad applicability. It is suitable for staining Nissl substance and neurons in paraffin-embedded tissue sections. The presence or disappearance of Nissl bodies is a crucial indicator of neuronal damage. Nissl bodies may dissolve or even disappear under pathological conditions such as encephalitis, cerebral ischemia, and axonal reaction. This reagent is for research use only and not intended for clinical diagnosis or other purposes.
Materials to Be Prepared by User
1. Graded ethanol, distilled water
2. Incubator, alcohol lamp, microscope
Procedure (for reference only)
1. Fix fresh tissues in ethanol, Carnoy's fixative, or 10% neutral formalin solution, followed by routine dehydration and embedding.
2. Cut sections with a thickness of 6–8 μm, then perform routine dewaxing to water using xylene or environment-friendly dewaxing clearing solution.
3. Immerse the sections in Cresyl Violet Stain, place the staining jar in an incubator and incubate at 56℃ for 1 hour or at 37℃ for 2–3 hours. If the staining result is unsatisfactory, heat the sections over an alcohol lamp until bubbles appear (approximately 10 minutes), then rinse with cold distilled water.
4. Differentiate the sections in Nissl Differentiation Solution for 1–3 minutes, and observe under a microscope until the background is nearly colorless.
5. Dehydrate rapidly with anhydrous ethanol, clear routinely with xylene or dewaxing clearing solution, and mount with neutral mounting medium.
Staining Results
Nissl bodies: Purple
Background: Nearly colorless or pale blue
Precautions
1. Nissl bodies are prone to dissolution after being isolated from the body. Therefore, tissues must be fixed immediately after collection; otherwise, effective staining cannot be achieved.
2. Tissue fixation plays a vital role. Ethanol, Carnoy's fixative, or neutral formalin solution can be used as fixatives.
3. This staining solution yields favorable results for Nissl staining of paraffin-embedded tissue sections.
4. Prepare paraffin sections with a thickness of 6–8 μm or 25 μm (25 μm-thick sections are required for evaluating the density of cortical neurons).
5. Stained specimens must be stored in the dark to prevent fading.
6. For your safety and health, please wear a lab coat and disposable gloves during operation.
7. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
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