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 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 levels, 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 vary across different neuronal subtypes. 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 key sites for protein synthesis in neurons, their volume decreases significantly when neurons are stimulated.
Nissl Staining Solution (Methyl Violet Method) is characterized by simple operation, stable staining results, short differentiation time, 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 critical indicator of neuronal damage. Nissl bodies may dissolve or even disappear in 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. Neutral formalin solution, distilled water
2. Microscope
Reference Procedure (for reference only)
1. Fixation: Fix tissues using ethanol, Carnoy's fixative, or neutral formalin solution, followed by routine dehydration and embedding.
2. Tissue Sectioning: Prepare paraffin sections with a thickness of 7-10 μm or 25 μm (see Precaution 5). Dewax the sections to water using xylene or environment-friendly dewaxing clearing solution.
3. Stain the sections with Methyl Violet Staining Solution for 10-20 minutes, then rinse with distilled water.
4. Differentiate the sections with Nissl Differentiation Solution for 4-8 seconds until most of the background staining is removed.
5. Dehydrate directly through anhydrous ethanol, then clear with xylene or dewaxing clearing solution, and observe under a microscope.
6. Repeat step 3 if necessary; apply a small amount of Nissl Differentiation Solution for re-differentiation after re-staining.
7. Rinse thoroughly in xylene or dewaxing clearing solution, then mount the sections with Canada balsam or DPX mounting medium.
Staining Results
Nissl substance or Nissl bodies: Purple-black-blue
Neurons: Pale purple-blue
Cell nuclei: Purple-blue
Precautions
1. Nissl bodies are prone to dissolution post-mortem. Tissues must be fixed immediately after dissection; otherwise, Nissl bodies cannot be stained effectively.
2. Tissue fixation plays a crucial role in staining success. Ethanol, Carnoy's fixative, or neutral formalin solution are recommended fixatives.
3. This staining solution yields optimal results for Nissl staining of paraffin-embedded tissue sections.
4. To confirm the presence of Nissl substance, differentiation with Nissl Differentiation Solution is mandatory after staining.
5. Prepare paraffin sections with a thickness of 7-10 μm or 25 μm (25 μm-thick sections are required for evaluating the density of cortical neurons).
6. Stained specimens must be stored in the dark to prevent fading.
7. For your safety and health, please wear a lab coat and disposable gloves during operation.
8. Please use the reagent as soon as possible after opening to avoid compromising subsequent experimental results.
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