User:Nyanez1/sandbox/Nerve Staining

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.

=History=

=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.

Sihler's Method
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..

Strengths
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.

Weaknesses
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.

Nissl Method
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 rough endoplasmic reticulum] has [[ribosome|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.

Strengths
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.

Weaknesses
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.

Bodian Method

 * (Explanation on how method involves Protargol (of which must include the trademark thing) and how it helps on the staining of nerve fibers in tissue)

Holmes Silver Nitrate Method

 * (nerve fibers, neurofibrils in tissue section)

Bielschowsky Method

 * (Explain: demonstration of nerve fibers, neurofibrillary tangles and senile plaques in Alzheimer's disease)
 * (Add usage second procedure similar to this, PAS)

Sevier-Munger Method

 * (similar to Bielschowsky, must state and find article on why it's different exactly)

Mallory PTAH Method

 * glial fibers

Holzer Method

 * glial fibers

Cajal (gold sublimate) Method

 * glial fibers

Weil Method

 * myelin

Luxol Fast Blue (stain) Method

 * myelin

Weaknesses
=Combinations of Staining Methods=

Possible future uses of methods
=References=