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Nerve staining is the process of which a nerve, or a set of nerves, is darkened or highlighted compared to surrounding areas in order to show differences or a certain process going on in the nerve.
There are several types of nerve staining, some used more than the others with different levels of success and difficulty. Each type of staining process has advantages and disadvantages for these reasons, being used for different types of research as well as to study different parts of a neuron, a nerve, or a set of nerves at the same time.
- 1 History
- 2 Types of Procedures
- 2.1 Golgi Method
- 2.2 Sihler's Method
- 2.3 Nissl Method
- 2.4 Bodian Method
- 2.5 Holmes Silver Nitrate Method
- 2.6 Bielschowsky Method
- 2.7 Mallory PTAH Method
- 2.8 Holzer Method
- 2.9 Cajal (gold sublimate) Method
- 2.10 Weil Method and Luxol Fast Blue (stain) Method
- 3 Combinations of Staining Methods
- 4 References
It was Camillo Golgi who started studies on human nerve cells during the end of the 19th century and tried to find a way to stain the cells by means of introducing metals when Golgi was chief medical officer in a psychiatric hospital. After the introduction of the neuron doctrine by Santiago Ramón y Cajal - a theory for which Golgi disagreed completely and wanted to pass along the "reticular doctrine" - research started to get stronger on the field of nerve staining, thus leading to the now more sophisticated methods that still include the staining introduced by Golgi and Cajal.
Types of Procedures
Depending on the research wanted and the extent of information wanted to gather, these following methods are the best known and most used for nerve research.
Camillo Golgi came up with this method when doing the first research on nerve staining, and called this method the "black reaction" due to the attachment of silver chromate to the neuron membrane, the chemical produced from the combination of silver nitrate and potassium dichromate. This procedure helped identify the neurons in such a way that eventually the proposed neuron doctrine by Golgi was accepted  .
The main strength, and accomplishment, of this method is the capacity to see the main parts of the neuron, thus showing that the nervous system is not just one continuous loop but composed of many cells.
This method is not as good as was thought back in its days, and thus it has been modified many times to get better results. Now, research has gone to the extent of wanting to stain particular organelles than rather the entire cell.
Charles Sihler first introduced this method to brighten up nerve endings in snakes and frogs. However, forgotten by many years, this method was used and modified by several doctors, finally to be used in current methods of nerve research with the implementations given by Liem and Douwe van Willigen on the last half of the 20th century.
Sihler's method consists of several parts which must be done within a period of 3-4 months :
- Fixation: This part is done in order to make sure that the cells are prepared for proper staining. This is usually done with an amount of formalin solution(in most cases, it's a 10% unneutralized formalin solution .
- Cell Maceration and Depigmentation: The cells must go through a solution in order to lose its steady, solid state and "soften" up so the stain can come in. Adding to this, the cells must also be depigmented in order for the stain to be visible. This process takes about a month in processing, and the solution must be changed daily or every other day. The solution used could be a solution containing potassium hydroxide solution with hydrogen peroxide  .
- Decalcification: This part of the process makes the cells even more flexible, taking away the last chemicals that would make the cell rigid. This would be done with Sihler's solution I (1:1:6=glacial acetic acid:glycerin:1% aqueous chloral hydrate solution). The samples would have to be soaked in this solution for 4 weeks and refreshed every week  .
- Staining: Sihler’s solution II, which is a 1:1:6=Ehrlich’s hematoxylin:glycerin:1%-aqueous-chlorate-hydrate solution, is applied now to colorize the sample. Depending on the sample size and tissue density, this part of the process might take between 3 to 4 weeks  .
- Destaining: This involves using Sihler’s solution I again, with some stirring in the part of the process. This is done to the point in which the nerve twigs are revealed. The only way to get this to perfection depends on the specimen size and the experience of the technician  .
- Neutralization: Depending on the size of the sample as well as the density of the tissue, the next step involves rinsing water and 0.05% lithium carbonate in order to neutralize the acidic state of the sample. Usually each rinse tends to take at least half an hour  .
- Clearing: The last part involves washing the sample with increasing concentrations of glycerin (starting with 40%, ending with 100%, each concentration being increased 20%) each day until overstained regions are washed out.   .
This procedure has four main advantages. One of them has to be the ability to see finer, smaller nerve twigs without cutting open tissue. This helps on not breaking tissue and the possibility of breaking these nerves as well when doing manual dissection. The second and probably the most important advantage is the visualization of the nerves to the naked eye of the structure of the nerve pattern in the sample. This adds to the next point, which is that this technique allows us to see adjacent structures to the nerves and how do they function in conjunction to them. Adding to this, both the innervation pattern of the nerves and the arterial distribution inside of them can be seen when using this technique  .
Given that the technique is capable of staining the myelin sheath around the axon only, it could be a motor or sensory neuron, so one would not be capable of distinguishing this unless looking at the main branch of the nerve. As seen in the description of the technique, this technique is very time consuming, adding also that the experience of the technician is very important in this procedure. As said previously, most of the procedure is basically heavily dependent on the sample size and the density of the tissue being experimented on; due to these two variables, staining quality may be very different.
This method, invented by German psychiatrist Franz Nissl, bases itself on staining the Nissl body, rough endoplasmic reticulum granules that are present in neurons, mainly found in the dendrites and soma. The [[endoplasmic reticulum|rough endoplasmic reticulum] has ribosomes that allow the stain to work on them. The stain is a cresyl echt violet solution, which acts on the ribosomes, making a basic solution and turning them from blue to purple. Depending on the procedure and how basic the solution is, aside from the Nissl bodies, the nuclei of the cells in the sample may be stained at the same time.
The method is widely used in staining neurons in the spinal cord and the brain, as well as glia and blood vessels, giving a good view of how each part works along with the other.
There might some time consumption on trying to repeat the addition of the balsam-xylene solution before checking on the microscope, but it is substantially small compared to other methods, such as Sihler's method.
This method involves using a silver proteinate called Protargol™ that along with some copper create a chemical reaction. This causes the staining of of nerve fibers in tissue sections. This method is best used for staining in peripheral nerves or samples of the cerebral cortex. Hydroquinone is the reducing reagent in the procedure, which must be used under a hood due to its fumes being harmful to humans and the environment
The main strength about this method is the difference in coloring given due to the reaction. The results of the procedure will give you the nuclei of the cells looking black, the background looking light gray or blue, and the nerve fibers looking black.
The most clear one is the usage of hydroquinone, which is harmful and must be used discretely. Although this chemical can be controlled, its use is a clear weakness, possibly being a potential carcinogen. This procedure does not necessarily given constant results.
Holmes Silver Nitrate Method
- (nerve fibers, neurofibrils in tissue section) Holmes came with this method thinking of modifying Bodian's procedure, saying that the inconsistencies found were mainly because the acid used in the Bodian method never truly neutralized. Thus, Holmes thought of using a buffered impregnated solution, preferably a 10% neutral buffered formalin solution  .
This method is good for staining section of the cerebral cortex for the demonstration of nerve fibers and neurofibrils. The sections can be between 10 and 15 μm  . The coloring for the axons, nerve fibers, and neurofibrils in the cells will be black.
Although it is not a huge problem if one were to focus on the parts to be stained, a problem with this procedure would be the impracticality of not staining the nuclei and differentiating the different parts of the cells.
The method consists of a silver nitrate combination that will be toned with gold chloride. The method allows us to demonstrate nerve fibers, is mostly useful in the central nervous system and it specifically helps show the presence of neurofibrillary tangles and senile plaques, characteristic features in Alzheimer's disease. The axons are toned black while the rest of the bodies are toned gold  . The Sevier-Munger method is similar to this one, only that instead of consisting of a silver nitrate combination, the tissue is impregnated with an ammoniacal silver solution.
As previously announced, one of the main reasons for the development of this method was for the identification of certain characteristical features in the neurons of a patient with Alzheimer's disease.
The method helps on checking neurofibrillary tangles and senile plaques, but possibly not many other benefits can be gotten from this method after that.
Mallory PTAH Method
This method allows us to show glial fibers, and it particularly uses phototungstic acid with hemaelin (in a 20:1 ratio, respectively) to turn them blue due to the solution, while the rest would turn red or brown due to the excess acid .
The process is very straight forward compared to others. One thing to add is the dehydration steps for the cells are very fast, which helps on destaining the samples.
The main weakness with this method, as well as a couple of the upcoming ones is the fact that they are mainly used for checking a couple of particular organelles without seeing anything else. Although helpful when finding answers to particular questions on those organelles, scientists cannot work on the cells until they are destained . One thing that was wanted from this method is the possibility of checking areas of gliosis, for which it cannot be done with this method since the normal and abnormal astrocytes cannot be separated nor identified . Adding to this, some of the chemicals used in this method are a little toxic.
Compared to the Mallory PTAH method, the added objective aside from just showing glial fibers is to show the areas in tissue samples showing gliosis. Glial fibers are stained with crystal violet and are resistant to decolorization with the alkaline aniline-chloroform mixture used in this procedure .
The main strength of the procedure is the ability to detect gliosis. The abnormal astrocytes and processes are stained in the tissue, letting us see the areas where gliosis is found. This method also minimizes the use of toxic chemicals, with the process being completed in eight hours, fast completion compared to other methods.
Once again, the main weakness as the previous method is singularity of staining; meaning that the staining will only stain the specified and wanted area. This means that other wanted areas of staining would not stain and must go for another procedure.
Cajal (gold sublimate) Method
Astrocytes are selectively stained with the Cajal gold sublimate method on frozen sections in this method. Although is one of the first well-known methods, this method has been almost completely replaced by immunohistochemical procedures. It's better for use on samples of cerebral cortex.
At the start of nerve research and staining, this procedure was very useful and knowledgeable. A long time has passed since its introduction, changing the procedure and being almost obsolete. Still, at the moment of its introduction, it was one of the most important staining procedures of its time.
There are other methods that are better now, and they require less time with less chemicals. The science behind staining has progressed so much to the point that this method is almost obsolete.
Weil Method and Luxol Fast Blue (stain) Method
The two methods characterize themselves on the purpose of staining the myelin sheath, each being different from each other but still accomplishing the staining goal.
The two methods are good at staining the myelin sheath effectively, the Weil method staining it blue to blue-black while the luxol fast blue method to blue (usually the Weil method gives a darker stain than the luxol fast blue method) .
Combinations of Staining Methods
Given that certain methods accomplish staining on particular areas of neurons, some of these methods have end up being combined in order to accomplish bigger areas of staining and better views of the samples.
- "Introduction to Special Stain Techniques: Nerve Tissue Staining" (PDF). Histology Couse. Retrieved 9 December 2013.
- Zaqout, Sami (2012). "A Golgi Study of the Camel Cuneate Nucleus". The Anatomical Record. 295. Unknown parameter
- Won, Sung-Yoon (2011). "Clinical and anatomical approach using Sihler's staining technique (whole mount nerve stain)". Anatomy & cell biology. 44 (1): 1–7. doi:10.5115/acb.2011.44.1.1. Unknown parameter
- Yung, Hun-Mu (2013). "Sihler staining study of anastomosis between the facial and trigeminal nerves in the ocular area and its clinical implications". Muscle & nerve. doi:10.1002/mus.23875. Unknown parameter
- Peters, A. "Experiments on the Mechanism of Silver Staining" (PDF). Journal of Cell Science. Retrieved 10 December 2013.
- Manlow, A. (1992). "A non-toxic method for the demonstration of gliosis". Journal of neuropathology and experimental neurology. 51 (3): 298–302. Unknown parameter
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