Lab Chronicles: My Experience in the Histology Lab – Week 4, 5 and 6

Throughout my final weeks in the Histology lab, I performed a variety of special stains in order to detect connective tissues, carbohydrates, microorganisms, and pigments in the diagnosis of certain diseases. I also performed immunohistochemistry in the detection of cancer markers. In a routine hematoxylin and eosin stain, many of these components are not visible and therefore special stains are needed in order for them to be seen.

Connective Tissue Stains

There were several connective tissue components that I stained within these weeks. Connective tissue is responsible for connecting epithelium to the rest of the body and some connective tissues have specialized functions and diagnostic importance. The main connective tissue present in the body is collagen, which provides strength to surrounding structures. A stain used in the detection of collagen is the Trichrome stain. In the Gomori’s One-Step Trichrome stain, collagen and smooth muscle are differentiated. There are several steps involved in performing this stain. First, tissue slides are placed in Bouin’s solution (containing picric acid, formaldehyde and glacial acetic acid), which mordants the tissue and enhances the stain. Then, nuclei of the tissue are stained with Weigert’s Iron Hematoxylin. An iron hematoxylin is used for this stain because it resists any decolorization from the acidic reagents used in the following steps. Then, slides are stained with the Gomori’s One-Step Trichrome solution, which contains chromotrope 2R, light green, phosphotungstic acid and glacial acetic acid. The mechanism used in this stain is termed porosity, meaning that larger dye compounds only bind to large tissues, whereas small dye compounds bind to smaller tissues. Initially in staining the tissue, the chromotrope 2R (a small dye compound) enters all of the tissue (muscle, cytoplasm and collagen). It is then decolorized by the phosphotungstic acid present in the solution, removing it from tissues. Then, the light green dye (a larger dye compound) stains the larger tissues (collagen), and the chromotrope 2R stains the smaller tissues (cytoplasm and collagen) yielding a red color for cytoplasm and muscle fibers, and a green color for collagen fibers.

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Blood vessels demonstrated with Gomori One-Step Trichrome. Collagen appear as light green, whereas muscle appear as pink and red blood cells dark red.

Another common connective tissue present in the body is elastin. As its name suggests, it contributes to the elasticity of tissue. For the demonstration of elastin, the Verhoeff’s stain was performed. In the first step of the stain, tissue slides are stained in a Verhoeff’s solution that contains iron hematoxylin, 10% ferric chloride and iodine. The ferric chloride and iodine serve as mordants and oxidizers for the iron hematoxylin. When stained in this solution, all of the tissue present in the slide is black. The slide is then differentiated with a couple of dips in a 2% ferric chloride solution. This differentiation occurs because the 10% ferric chloride present in the Verhoeff’s stain moves towards the differentiating solution of a lower concentration. Then, the slide is counterstained in a Van Gieson stain that consists of acid fuchsin and picric acid. The acid fuchsin present in the dye stains the collagen red. The picric acid present in the dye stains the muscle and red blood cells present in the tissue yellow. Elastin present in the tissue will be stained black.

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Elastic artery demonstrated by Verhoeff’s Elastic stain. Elastin is shown as black fibers, collagen is stained red and red blood cells are stained yellow.

Reticulin is another connective tissue present in the body, and its function is to provide a supporting network for the basement membranes of tissues. The special stain used to demonstrate reticulin in tissue is the Gordon and Sweet stain, which uses a silver and ammonia solution (termed ammoniacal silver) to impregnate reticulin. First, potassium permanganate is added to the tissue slide in order to oxidize the carbohydrate components of the reticulin to aldehydes. Then, ferric ammonium sulfate is added, which sensitizes and binds to the tissue. The silver ions from the ammoniacal silver solution then impregnate the reticulin fibres and replace the sensitizer, becoming an insoluble silver salt. A reducer (formalin) is then added to the slide and reduces the silver salt to its metallic form. Gold chloride is then added to tone the reticulin and provide better contrast to the rest of the tissue. Excess silver and gold chloride is removed by sodium thiosulfate. Finally, a counterstain of either nuclear fast red or light green is applied to stain the background tissue and give better contrast to the slide.

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Hepatocytes in liver demonstrated in Gordon and Sweet’s stain. Reticulin fibers are stained black, and background is green.

The final connective tissue that I stained throughout this week was adipose tissue, more commonly known as fat tissue. As we all know, adipose tissue is responsible for storing and metabolizing fat in the body. The special stain used to identify adipose tissue is the Oil Red O stain. Unlike the previously mentioned stains, Oil Red O cannot be performed on tissues that have been fixed and processed, because xylene and alcohols present in the processing solutions dissolve fats. Therefore, an unfixed frozen section tissue is needed for staining. The procedure for Oil Red O is very simple and works by the method of selective solubility in which the dye is more soluble in the fat present in the tissue than its solvent, alcohol. First, the Oil Red O Solution is applied on the slide for an appropriate amount of time, and then stained in hematoxylin to demonstrate nuclei present.

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Steatosis of the liver (fatty liver) seen as red globules in the Oil Red O stain.

Carbohydrate Stains

There are also several clinically significant carbohydrates that are stained in the Histology laboratory. Glycogen, a polysaccharide found in the body, is one of the carbohydrates most commonly stained. The Periodic Acid Schiff stain is used to demonstrate this carbohydrate, along with other neutral polysaccharides and basement membranes. The first step of the stain is to apply the periodic acid to the tissue slide. This acid oxidizes the glycogen (or other polysaccharides) present in the tissue to aldehydes. Then, Schiff’s reagent is added to the slides that forms a magenta colored complex at the aldehyde groups. Schiff’s reagent is created by removing its color using sulfurous acid. Color is regained when the reagent binds to the aldehyde groups present in tissue. Further washing of slides in water causes the recolorization of the stain to create a magenta color. A hematoxylin counterstain is added to stain nuclei.

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Glomerulus of kidney stained with Periodic Acid Schiff to demonstrate basement membranes (magenta).

Acid mucopolysaccharides (also known as glycoaminoglycans) are another carbohydrate tested in the Histology laboratory. This carbohydrate is commonly found in mucus and fluid surrounding joints. Inherited conditions, such as mucopolysaccharidoses, exist where mucopolysaccharides do not get broken down by the body and buildup in tissue. Excess acid mucopolysaccharides are also present in other conditions, such as mesothelioma. The Alcian Blue stain is used to stain these kinds of carbohydrates. Alcian Blue is a copper phthalocyanin basic dye and when combined with a 3% acetic acid solution (which brings it to a pH of 2.5), both sulfated and carboxylated acid mucopolysaccharides are stained blue. When the dye is combined with 0.1N hydrochloric acid (pH 1.0), only sulfated acid mucopolysaccharides are stained blue. Then, a nuclear fast red counterstain is applied to stain the background of the tissue red.

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Alcian Blue demonstrating acid mucopolysaccharides (blue) of the colon.

Another carbohydrate that is stained is acid mucin, which is secreted in inflammatory conditions and intestinal tumors. A stain used in the detection of this carbohydrate is the Mayer’s Mucicarmine stain, which is commonly used to identify adenocarcinomas of the gastrointestinal tract. First, hematoxylin is added to slides to stain nuclei. Then, a mucicarmine solution, made up of aluminum salts and carminic acid, is added to the tissue. These components form a positively charged complex that attracts and binds with acid mucins present in tissue, staining them red. Finally, tartrazine is added to stain the background tissue yellow.

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Acid mucins (red) in colon stained by Mayer’s Mucicarmine

Microorganism Stains

Many stains are performed in Histology for the identification of microorganisms. Some are shared with the Microbiology laboratory, such as the Gram stain for the identification of bacteria. In addition to the Gram stain, the Ziehl-Neelson stain for acid-fast bacilli is used, in the diagnosis of patients with tuberculosis. First, a carbol fuchsin is added which enters the mycolic acid of the acid-fast bacilli, resisting decolorization from subsequent steps with alcohol and staining them red. An acid alcohol solution is then added to decolorize the background staining present in the slide. Then, the background is lightly stained with methylene blue, in order not obscure potential acid-fast bacilli present in the tissue.

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Mycobacterium tuberculosis stained red in Ziehl-Neelsen with blue background

Tissues can also be infected with fungi and the Grocott’s Methenamine Silver stain can be used to stain these tissues. Similar to the Gordon and Sweet’s stain, it is another silver impregnation technique. First, the carbohydrates present in the fungi are oxidized to aldehydes with the help of chromic acid. Excess chromic acid is then removed with sodium metabisulphate. The silver solution is then added to tissues and becomes an insoluble silver salt. The aldehyde groups present reduce the silver salt to its ions. Gold chloride is then added to tone the silver and sodium thiosulfate removes any excess silver gold chloride, just as the Gordon and Sweet’s stain. A light green counterstain is then applied to stain background tissue and provide contrast.

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Fungal hyphae and Pneumocystis jirovecii (black) stained by Grocott Methenamine Silver

Pigment Stains

I also performed one common stain for clinically significant pigments in the Histology laboratory, Perls’ Prussian Blue. It is used to stain ferric ions present in tissue that are commonly found in hemosiderin, which is a byproduct of red blood cell death. First, the slide is treated with hydrochloric acid that releases the ferric ions from the hemosiderin. Then, potassium ferrocyanide is added and a single displacement reaction occurs yielding ferric ferrocyanide. Perl’s Prussian Blue is an example of a histochemical stain, as it is not the dye conferring color to the tissue, but rather the chemical reaction between the reagent (potassium ferrocyanide) and the element of interest (ferric ions) that produce a compound that yields a blue color.

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Ferric ions (blue) from hemosiderin in liver tissue demonstrated by Perls’ Prussian Blue

Immunohistochemistry

Throughout my last weeks in the Histology laboratory, I also performed immunohistochemistry, which is the detection of antigens in tissue using antibodies. It is used in a clinical setting to type tumors and detect cancer markers in the diagnosis of cancer. It can also be used to detect microorganisms in tissue, such as hepatitis viruses, cytomegalovirus and Helicobacter pylori. The principles of antigen-antibody binding are used in immunohistochemistry. Tumors and microorganisms present in tissues have antigens present that elicit an immune response. This immune response stimulated B lymphocytes to produce antibodies that bind to a specific epitope of the antigen.

In performing the immunohistochemistry procedure, slides must be prepared to incubate at 37°C overnight. Preanalytical factors that can hinder testing include delayed fixation, or even excess fixation, as both of these factors can cause a loss of antigenicity in tissues. The following day, antigen retrieval is performed on any formalin fixed tissues to remove the crosslinks of proteins that mask antigen epitopes. At the Histology laboratory, heat-induced epitope retrieval was performed in a heated buffer solution held at different pH levels to break the formalin crosslinks. If antigen retrieval is not performed on formalin fixed tissues, those tissues could be reported as falsely negative and important diagnostic markers could go undetected. After retrieval, blocking is performed using serum and/or hydrogen peroxide. Serum blocking is used to block polyclonal antibodies that yield non-specific reactions in immunohistochemistry. Hydrogen peroxide is used to inhibit endogenous peroxidases from creating falsely positive reactions. Afterwards, the primary antibody is added which binds to the antigen of interest in the tissue. This process occurs in a humidity chamber to prevent the tissue slides from drying. Then, a secondary antibody against the primary antibody is added that contains a detection system, most commonly enzyme labels, such as horseradish peroxidase. A chromogen is added, such as 3,3’-diaminobenzidene (DAB) or Nova Red in order to detect the antigen antibody reaction. DAB is demonstrate on the slide in a brown color. For this reason, it is not wise to use this chromogen in the detection of melanoma markers as melanin is brown. Therefore, Nova Red is used in this case. Between each of these steps, the slides are washed with buffer (either Tris-buffered saline or phosphate-buffered saline) in order to remove any antibodies that are not bound to the tissue, in order to prevent non-specific staining. Finally, the tissue slides are stained in hematoxylin to stain nuclei and provide a contrast in staining. Each slide was stained with a positive control tissue that is known to contain the antigen of interest.

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Mart-1 antibody (stained red) used in the detection of melanoma using immunohistochemistry