Golgi stain

Summary

The Golgi method is capable of detecting minute morphological changes in the dendrites and dendritic spines of nerve cells in the brain of animals caused by drug treatment or neurological diseases, and observing the differences in nerve development, death, and transmission.

Operation method

Golgi stain (Golgi-Cox method)

Principle

Golgi staining includes silver Golgi staining and mercury Golgi staining techniques. The principle of silver Golgi staining is that potassium dichromate solution soaked in brain tissue reacts with silver nitrate solution to produce a black precipitate of silver chromate, which is deposited in nerve cells due to the silversmithing nature of the tissue. Two main types of silver Golgi techniques are widely used: the rapid Golgi method and the Golgi-Kopsch method. Principle of Mercury Golgi Staining: Due to the shortcomings of silver Golgi staining, Cox modified the Golgi technique, which became known as the "Golgi-Cox method". A mixture of potassium dichromate and mercuric chloride is used, and potassium chromate is added to modulate the acidity of the solution.The chemistry of Golgi-Cox staining is not fully understood; the black deposits formed by alkalinization of Golgi-Cox-impregnated neuronal cells are thought to be complex oxides of black mercuric sulphide and some metals.

Materials and Instruments

Instruments:
3D scanner scanning or orthostatic microscope (with photo system)
Material:
Animal and postmortem human brain tissue
4% (w/v) paraformaldehyde, isopentane, dry ice
Acrylated violet solution
50%/75%/95%/100% Ethanol, xylene
Tablet sealer, blades, staining vat, cryo-holders
Gel-coated microscope slides, pipettes, filter paper
Coverslips, FD Rapid Golgi Staining Kit

Move

The procedure of this experiment is based on the optimized Golgi-cox technique:

1. Mix solutions A and B in the kit 24 hours in advance. 2;

2. Remove the mouse brain tissue and rinse it with clean PBS, then place it in the mixture of A and B, and store it at room temperature away from light. After 6 hours of immersion or on the following day, replace with new impregnation solution and store at room temperature and protected from light for 2 weeks (at least 2 mL of impregnation solution for each brain tissue). 3;

3. transfer the tissue to Solution C. After 24 h of immersion or the next day, replace with new Solution C and store at room temperature away from light for 7 days;

4. Vibratory slicer to prepare the brain tissue into 100~200 μm thick slices, transfer the samples to gelatin-coated microscope slides containing Solution C and dry at room temperature for 3 days. 5;

5. Rinse the slide twice with distilled water for 4 min each time. 6;

6. make a mixture (ready to use) of Solution D, Solution E and water in the ratio of 1:1:2 and place the slide in the mixture for 10 min. rinse the slide twice with distilled water for 4 min;

7. the sections were dehydrated in 50%, 75% and 95% ethanol for 4 min at each concentration gradient. 8. the sections were then placed in a freezer;

8. the sections were then dehydrated in anhydrous ethanol four times for 4 min each time. 9. the sections were placed in xylene in aqueous solution;

9. the sections were transparent in xylene 3 times for 4 min;

10. Seal the slices with sealer;

11. Image acquisition was performed by 3D scanner or ortho-microscope, and the second or third level dendrites were selected for quantitative analysis, and the number of dendritic spines was calculated by Image J software.

Caveat

1. Fresh or short-term fixed brain tissue is used for the samples in this experiment. Brain tissue that has been formalin-fixed or freshly frozen is not recommended. 2;2. large brain samples (e.g., rat brain tissue) should be cut with a sharp blade into pieces approximately 10 mm thick;3. the mixture of Solution A and Solution B should be prepared at least 24 h in advance without stirring;4. in step 2, the tissue container can be gently vortexed from side to side (do not shake!) for a few seconds twice a week during the brain tissue immersion. The tissue container can be gently swirled (no shaking!) from side to side for a few seconds twice a week during step 2;5. solutions A and B contain heavy metals that are toxic in contact with skin and potentially fatal if swallowed. Always wear protective clothing, gloves and other protective equipment when performing experiments, and do so in a chemical fume hood. Waste of solutions A and B should be collected and disposed of by a specialized department. 6;6. Sections stained with Golgi should be stored away from light.

Common Problems

1. Dark background color:

Possible reasons are that solutions A and B were not configured 24 h in advance or that the brain tissue was immersed in the A and B mixture for too long, more than 14 days.

2. negative results:

This may be due to perfusion of the animal before removing the brain for the experiment.

3. Golgi staining is not dark:

This may be due to the fact that the brain tissue was not stained at all in the A and B mixture for less than 14 days or because there was too little fluid to completely infiltrate the brain tissue or the reagents were out of date.

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