Tissue Staining

Staining Tissue Definition

Stains or dyes allow us to examine structures in more detail by enhancing the contrast of the structures of the cell, and they can also go through cell walls helping researchers analyze metabolic processes.

We usually look at these structures under microscopes after staining. This is because tissues are transparent to our eyes without stains, so they must be stained or colored with different dyes. Stains and dyes are usually used commercially in the lab to learn about biological tissues and Cells and diagnose diseases.

Stains are usually made of organic salts with positive and negative ions. Ions are charged molecules.

• Basic stains have positively charged cations, allowing them to bind to negatively charged structures such as nuclear components. An example of a basic stain is hematoxylin.

• Acidic stains contain negatively charged anions, allowing them to bind to positively charged structures such as components in the cytoplasm. An example of an acidic stain is eosin.

Staining Tissue Structures

You can think of stains as working with hair dyes in the salon. The hair dye penetrates the cuticle of your hair and bonds with it. This causes the hair to be permanently colored like cells and tissues when dyed with stains.

Similar to hair dyes, different types of stains color different cell structures! For example, some stains can dye the entire cell, while others stain specific compartments such as nuclei.

• The different dyeing potential of stains is why we often use multiple dyes together.

When we stain human tissues, we stain four basic types: muscle, connective, epithelium, and nervous tissue.

• Muscle tissues are tissues that are located on The Heart's walls (cardiac), attached to tendons or bones (skeletal), and hollow internal organs (smooth).

• Connective tissues are tissues found in between other tissues throughout the body.

• Epithelium tissue is the tissue that lines the walls or cavities of many organs. This tissue also includes our skin or epidermis.

• Nervous tissue is the tissue that comprises the nervous system. The nervous system allows the brain and spinal cord to communicate with the body.

The four basic tissues we usually study for Plants are dermal, vascular, ground, and meristematic.

• Dermal tissue is also called the epidermis, the plant's skin. It covers the plant's leaves, flowers, roots, and stems.

• Ground tissue is the tissue that's neither dermal nor vascular (parenchyma, collenchyma, and sclerenchyma).

• Vascular tissue is the tissue that transports nutrients and fluids (the Xylem and Phloem).

• Meristematic tissue consists of cells that aren't differentiated that can undergo Cell Division and develop into other tissues.

We usually classify staining as direct or indirect. Where indirect staining involves a mordant while direct staining doesn't. One of the most well-known fields we use staining in labs is histology.

• Histology is the branch of biology that studies the structural anatomy of tissues. In contrast, cytology analyzes the structure of tissues, and we call it organology for organs.

Tissue Staining Types

There are a lot of stains in tissues, and we often combine them to visualize multiple parts of a cell or tissue. Here are some of the most common stains that scientists use:

Hematoxylin is a basic dye that stains acidic structures with a purple/blue hue, such as nuclear components, as shown in Figure 1. Nuclear components include the nucleus, where DNA is stored, RNA in ribosomes, and RNA in the rough endoplasmic reticulum.

Figure 1: Hematoxylin and Eosin stain (H&E stain) of brain tissue from a rabies encephalitis patient. Public Health Image Library (PHIL), CDC.

Eosin is a counterstain used after hematoxylin and stains with a pink/red hue, as shown in Figure 1. It's an acidic stain that targets basic structures such as the cytoplasm. The cytoplasm is material inside the cell that includes all cell structures except the nucleus.

Gram Staining

Gram staining is an indirect staining that uses iodine as a mordant. This type of staining is used to differentiate bacterial species by staining the cell wall.

Gram-positive Bacteria have thick peptidoglycan layers in their cell walls, causing them to retain the crystal violet stain. This means Gram-positive bacteria will be stained violet or purple, as shown in Figure 2.

• Bacillus anthracis, as illustrated in Figure 2, is a Gram-positive bacterium that are rod-shaped and cause anthrax. Anthrax results from spores getting into our bodies and result in severe illness.

Figure 2: Positive Gram stain with Bacillus anthracis. Public Health Image Library (PHIL), CDC.

In contrast, Gram-negative bacteria have thin peptidoglycan layers in their cell walls, causing them to stain pink/red instead, as shown in Figure 3.

• Shigella dysenteriae, as illustrated in Figure 3, is a rod-shaped Gram-negative bacteria that cause shigellosis. Shigellosis is an infectious disease that causes fevers, diarrhea, and severe stomach problems.

Figure 3: Gram-negative, rod-shaped, Shigella dysenteriae bacteria. Public Health Image Library (PHIL), CDC.

Tissue Staining Procedure

Before we can stain the tissues, the tissue samples must be prepared. The steps are generally fixation, processing, embedding, sectioning, and antigen retrieval if necessary.

Fixation is done to preserve the cell's shape and structure and to provide it with more rigidity using chemicals. Most people use the chemical 10% neutral buffered formalin for 24-72 hours for most samples. The other steps are usually automated in most labs nowadays.

Dehydration is the process that takes out water from the tissue to harden it. This step is most likely automated in a lab.

Embedding is the process of putting the tissues in paraffin wax to allow for better extraction of the tissues. You can see the result of embedding when you look at the suspended block of paraffin pictured in Figure 4.

As shown in Figure 4, sectioning with a microtome cuts the paraffin wax blocks from embedding into thin sections onto microscope slides for examination. We can think of the non-automated microtome photographed in Figure 4 as a pencil sharpener and the block of wax positioned near the top as your pencil. We turn the handle on the right side to get "pencil shavings." These pencil shavings are what we float on the water bath to place onto clean slides.

Figure 4: How scientists cut tissues. Wikimedia, EPA (Public Domain).

Antigen retrieval is when the chemical modifications done by formalin fixation are lessened or removed.

Once tissues are adequately prepared, they are ready to be stained. The general procedure depends on the stain being used. But in general, the steps to stain tissues are as follows:

• After sectioning or cutting tissues into thin slices of wax, as shown in Figure 4, we need to use a warm water bath to allow the tissue or thin sections of wax to float to the top, where we can collect them using the clean slides.

• Next, stain the slide with the chosen stain as needed. Make sure to follow the instructions for the specific stain you are using.

• Usually, if people have more than one slide to stain, they'll use staining racks and dip all of it back and forth into the stain solution and a water solution. The water solution is there to get rid of excess stains. Make sure to follow the instructions unique to each type of stain.

• Carefully wipe the back of the slide with paper towels.

• Now, you can get your slide onto your microscope sample side up and set the objective to 10X. 10X is the lowest magnification and gives you the most general view. After this, select your area of interest in the tissue and adjust the objective accordingly as illustrated in Figure 5.

Figure 5: Scientist examining a tissue slide under a microscope. The U.S. Air Force (Public Domain).

Tissue Staining Example

Examples of tissue staining or case studies in labs often include stain kits with multiple stains at once, such as Masson's trichrome stains.

In general, muscle and keratin appear red and pink for the cytoplasm, blue/green for collagens and bone, and black/brown for cell nuclei.

Scientists conducted a study where mice inhaled p-Chloro-α,α,α-trifluorotoluene, a carcinogen, for two years to learn about the toxicology and carcinogenic effects. In Figure 6, you can see a region of the lungs where there's a lack of blue/green fibrous connective tissue in a male control mouse.

When Figure 6 is compared to Figure 7, you can see extensive blue/green fibrous connective tissue because of peribronchiolar fibrosis caused by inhalation of the carcinogen. Basically, this study shows that scientists can use many different stains, such as the trichrome, to obtain necessary research or information regarding their experiments.

Figure 6: Masson's Trichrome Stain in a male mouse's bronchiole region in the lung. National Library of Medicine, National Institutes of Health.

Figure 7: Peribronchiolar Fibrosis in the bronchioles of the lungs of a non-control mouse. National Library of Medicine, National Institutes of Health.