Nippon staining assay

Summary

Source : Practical Laboratory Techniques in Neurobiology

Operation method

basic program

Principle

Nissl Staining is a method of staining nerve tissue with a basic dye. Nissl is an alkalophilic substance in the cytoplasm, which is widely seen in all kinds of neurons, and the shape, size, and number of Nissl in different neurons are different. The alkaline dyes used for Nichols staining are mainly tar violet, methylene blue, toluidine blue and sulfur cordial, etc. We mainly use tar violet and methylene blue. In our laboratory, we mainly use tar violet and sulfur cordial staining. The Niehl's staining method can stain Niehl's bodies, which can be used to observe the cellular structure of neurons; it can also be used to understand the damage of neurons through the observation of Niehl's bodies after Niehl's staining.

Materials and Instruments

Neuron
Tar violet Corydalis sulfur

Move

(1) Paraffin or frozen sections were sequentially subjected to xylene, anhydrous ethanol, 95% alcohol, 80% alcohol, 70% alcohol and distilled water, and the respective times can be referred to HE staining.

(2) 1% tar violet or 1% sulfur cordial staining for 10min to 1h.

(3) Distilled water washing.

(4) 70% alcohol color separation for several seconds to several minutes.

(5) Sequentially through 70% alcohol, 80% alcohol, 95% alcohol, each 2min dehydration.

(6)Anhydrous ethanol 2 times, each 5min. xylene 2 times, each 10min.

(7) DPX sealing. Nitrosomes were purple, nuclei were lavender

Caveat

(1) Sections should be dewaxed first in xylene if they are paraffin sections.(2) The staining time of tar violet or sulfur cordial is also flexible according to the newness of the dye preparation time, the thickness of the section and the ambient temperature.Reagent preparation：

（1） Tar violet solution 1g tar violet dissolved in 100ml distilled water, dissolved and filtered for use.(2) Sulfur cordial solution 1g sulfur cordial dissolved in 100ml distilled water, dissolved and filtered for use.

Common Problems

(1) The degree of color separation should be observed under the microscope, so that the nidus is dark and the dorsum is essentially colorless is optimal. If the color separation is not enough, it can be returned to the alcohol and color separation again. If the color separation is excessive, it can also be returned for re-dyeing.

(2) Niehl's staining should be sufficiently dehydrated, otherwise it is easy to fade.

(3) Although the HE staining method can also observe basophilic granules in the cytoplasm, the structure is not very clear, and the axons and dendrites of neurons are difficult to recognize. In Nissen staining, the Nissen bodies were clearly recognizable, and it was easy to distinguish between axons and dendrites. In Niehl's staining, Niehl's bodies are blocky (like tiger spots) or granular after staining, Niehl's body granules around the nucleus are larger, and near the edge of the smaller and elongated, such as in physiological conditions, Niehl's bodies are large and numerous, reflecting that the nerve cells synthesize proteins in a more powerful way, and the number of Niehl's bodies can be reduced or even disappeared in neuron damage, and the experimenter can choose between HE staining and Niehl's staining according to the purpose of the experiment.

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