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Basic histological staining methods

Introduction to the main staining methods used in histology.

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Hey everyone! This is Joao from Kenhub, and in this tutorial, we're going to look at some of the most common staining techniques we use in histology.

As you probably know, histology is the study of the microscopic anatomy of cells and tissues. So we used staining methods to visualize and distinguish the different parts of cells and tissues since cells and their structures are usually transparent or colorless. The types of dyes used to color cells and their components can either be specific to particular structures, chemical groups or even molecules, and it can also be non-specific in which case most of the cell is stained in the same way.

In this tutorial, we will cover some of the most common types of dyes used in histological staining of cells and their structures. Before we look at some examples of histological dyes, I want to give you a brief overview of the types of dyes that are used.

When staining tissue samples, dyes that are used are either acidic or basic or a combination of the two. And why is that, you might be asking. Well, cellular structures such as nucleic acids or proteins have charged groups which are known as phosphate groups or carboxyl groups, just to name a couple. The dyes used in histology are colored organic compounds which also have a charge. Acidic dyes carry a negative charge and so they bind to positively-charged cell structures. To give you a better idea of this, let's look at this quick visual.

Here we see the negatively charged dye binding to the positively-charged cytoplasmic proteins. This makes them appear colored – in this case, pink. Basic dyes, on the other hand, carry positive charges and as such will bind to negatively-charged cell structures. Here we see the positively-charged dye binding to the negatively charged cell membrane. This makes the structure appear blue in this example. Note that the neutral dyes can also be used and these don't carry any charge. I won't go into detail about the different types of basic or acidic or neutral dyes. This is just to give you a bit of a background into why these dyes bind to particular cellular structures.

Now let's move on to look at a few brief examples of histological dyes that are used to visualize cellular structures. Note that in this tutorial, we will look at a few of the most common dyes used in the histology namely haematoxylin and eosin, periodic acid-Schiff reaction, silver impregnation, toluidine blue, Masson's trichrome, and osmium tetroxide.

The first type of staining that we will look at is haematoxylin and eosin stain. This type of dye is often simply referred to as H&E stain. Here we see a micrograph showing skeletal muscle stained using this technique in order to visualize the tissue under a light microscope. As you can see, the muscle tissue looks pink in appearance and the purple dots you see distributed throughout the tissue are nuclei. Haematoxylin and eosin staining is one of the most commonly used staining techniques in histology. H&E staining uses two dyes – haematoxylin and eosin. Haematoxylin is a basic dye that stains acidic structures giving them a blue or purple appearance such as the nucleus. Eosin is an acidic dye that binds to basic structures such as intracellular membranes, cytoplasmic proteins and so on, and it will stain them in pink.

We are now moving on to the next staining technique we have on our list which is periodic acid-Schiff reaction. This is another common technique used in histology to add contrast to cells. Periodic acid-Schiff reaction is commonly abbreviated as PAS staining or PAS staining. This staining technique is used to stain carbohydrates and carbohydrate-rich molecules in cells giving them a deep red color. This technique is also used to aid in the diagnosis of many medical conditions.

For example, here we see a micrograph of a tissue sample from a patient stained using PAS staining method to show gastric signet ring cell carcinoma. Adenocarcinoma secrete neutral mucins which are carbohydrates and can therefore be identified using this staining method because the mucin is stained as a reddish-purple color or magenta while the nuclei are stained blue as you can see here. This type of staining also gives contrast to structures such as basement membranes or the mucin produced by goblet cells.

Now, let's quickly have a look at the next technique called silver impregnation. Silver impregnation also known as the Golgi method or silver staining is another technique used in histology to make cells visible under the microscope. This method is used to stain nervous tissue. Here you can see a micrograph showing a pyramidal cell or neuron that has been stained using this method so it can be visualized under a light microscope. This method which uses potassium dichromate and silver nitrate results in the deposition of silver chromate which is a brown-red crystal compound that we can see here. This happens in nervous tissue allowing their structure to be easily visualized under a light microscope. The structures of nervous tissue appear black as you can see in this micrograph or brown or even sometimes gold.

We're going to move on to another type of staining. This one is known as toluidine blue. The toluidine blue stain is used to stain nucleic acids and polysaccharides. It is a basic stain and therefore stains acidic structures. For example, nuclei appear blue when stained while polysaccharide-containing structures such as cartilage appear purple when stained. Here we see a micrograph stained using the toluidine blue staining method that shows a vasculitic peripheral neuropathy. But don't worry at this stage if you don't understand what that is. What you need to know is that this type of dye stains nucleic acids blue while staining polysaccharides purple.

Moving on to the next staining technique on our list which is known as Masson's trichrome. This next type of histological stain known as Masson's trichrome has one what may call a triple effect. This staining essentially results in three different contrasts. For example, with this staining technique, basophilic structures such as nucleic acids are stained blue, collagen is usually stained green or blue, and structures such as cytoplasm, erythrocytes, keratin and muscle are stained red. This micrograph here is an example of a sample stained using this method. It is taken from the skin of a mouse and as you can see, the muscle which is pointed here and here, erythrocytes that are circled in the center, and cytoplasm here are all stained in red. You can also see the circled nuclei are stained blue. You can then see that the collagen fibers on the left side of the slide are stained blue as well.

Last on our list of staining techniques, we have osmium tetroxide. Another type of staining technique used in histology is osmium tetroxide. It is used to stain structures such as fat, myelin and the Golgi complex. Although this dye is good for staining fats, it is also quite toxic so strict protocols are used in the lab when working with osmium tetroxide. Here we see an electron micrograph of plant tissue that has been treated with osmium tetroxide in order to visualize membrane lipids and vesicals. The osmium tetroxide binds to the phospholipid heads and plasma membranes. And this technique is usually used in transmission electron microscopy and scanning electron microscopy.

So far, we have covered a very small portion of the types of stain we used in histology. There are obviously more stains that can be used that are specific to certain cellular structures and their cellular composition like these examples. These stains mentioned here are just to give you a brief introduction into how different types of stains give different contrasts to different parts of the cells. The main take-home message is that in histology, we make use of various tapes of dyes that are used to stain cellular structures according to their chemical properties which allow us to visualize and differentiate these microscopic structures.

I hope you enjoyed this video and learned something about the wonderful world of histological staining. I will see you on the next tutorial.

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