Principle of experimental method
Nissl staining is a method of staining nerve tissue with basic dyes. Nissl bodies are a kind of basophilic substance in the cytoplasm and are widely found in various neurons. The shape, size and number of Nissl bodies in different neurons are different. The basic dyes used for Nissl staining are Cresyl violet (tar violet, cresyl violet, gram violet); thionine (thionine); tolridine blue (toluidine blue) and so on. Nissl staining can stain Nissl bodies to observe the cellular structure in neurons; it is also possible to understand neuronal damage by observing Nissl bodies after Nissl staining.
Franna Nissl (1860-1919) founded the Nissl staining method in 1892, famous for discovering Nissl bodies and Nissl degeneration.
The neuronal cell body includes a large nucleus with a ruffled nuclear envelope, sparse chromatin, and a prominent nucleolus. The cytoplasm in the cell body is Nissl’s granules, basophilic granules that represent the rough endoplasmic reticulum and give rise to the characteristic speckled basophilic appearance in many neurons. Nissl particles can be displayed with a number of stains such as neutral red, methylene blue, toluidine blue, and methyl violet. Variation in staining, pH, and timing of differentiation allow some stains to both highlight Nissl material only, as well as to show neuronal nuclei and glia. All kinds of nerve cells contain Nissl bodies, but their shapes, numbers and distribution locations are often different. Nissl bodies are also present in dendrites, but not in axons and in the axons of the soma. Nissl body changes due to changes in physiological state. Nissl body is an important part of protein synthesis in neurons. When neurons are stimulated, Nissl body in the cell body is significantly reduced.
tar violet, thionine
(1) Paraffin or frozen sections were stained with xylene, anhydrous ethanol, 95% alcohol, 80% alcohol, 70% alcohol and distilled water in sequence. The respective times can be referred to HE staining.
(2) 1% tar violet or 1% thionine staining for 10min to 1h.
(3) Wash with distilled water.
(4) 70% alcohol color separation for several seconds to several minutes.
(5) Dehydration through 70% alcohol, 80% alcohol, and 95% alcohol in sequence for 2 minutes each.
(6) 2 times of absolute ethanol, 5min each time. Xylene 2 times, each 10min.
(7) DPX coverslips. Nissl bodies are purple and the nucleus is lavender.
(1) If the sections are paraffin sections, they should be dewaxed in xylene.
(2) The dyeing time of tar violet or thionine is also flexibly controlled according to the old and new preparation time of dye solution, slice thickness and ambient temperature.
(1) Tar violet solution 1 g of tar violet was dissolved in 100 ml of distilled water, and filtered for use after dissolving.
(2) 1 g of thionine solution was dissolved in 100 ml of distilled water, and filtered for use after dissolving.
(1) The degree of color separation should be observed under the microscope, so that the Nissl corpuscle is dark and the background is basically colorless is the best. If the color separation is not enough, you can return the alcohol to separate the color again. If the color separation is excessive, it can also be returned for re-dyeing.
(2) Nissl staining should be fully dehydrated, otherwise it will easily fade.
(3) Although the basophilic granules in the cytoplasm can also be observed by HE staining, the structure is not very clear, and the axons and dendrites of neurons are difficult to identify. Nissl bodies are clearly identifiable in Nissl staining, and axons and dendrites are easily distinguished. In Nissl staining, the Nissl bodies are massive (like tabby) or granular after being stained. The Nissl bodies are larger around the nucleus and smaller and slender near the edges. For example, under physiological conditions, the Nissl bodies are large. The large number reflects the strong function of nerve cells to synthesize proteins. When neurons are damaged, the number of Nissl bodies can be reduced or even disappeared. The experimenter can choose HE staining and Nissl staining according to the purpose of the experiment.